Biopreservation by Lactobacillus Paracasei in Coculture with Streptococcus Thermophilus in Potentially Probiotic and Synbiotic Fresh Cream Cheeses

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Journal of Food Protection, Vol. 70, No. 1, 2007, Pages 228–235 Copyright ᮊ, International Association for Food Protection

Research Note Biopreservation by Lactobacillus paracasei in Coculture with Streptococcus thermophilus in Potentially Probiotic and Synbiotic Fresh Cream Cheeses

FLA´ VIA C. A. BURITI, HAI´SSA R. CARDARELLI, AND SUSANA M. I. SAAD*

Departamento de Tecnologia Bioquı´mico-Farmaceûtica, Faculdade de Cieˆncias Farmaceûticas, Universidade de Saõ Paulo, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/1/228/1680018/0362-028x-70_1_228.pdf by guest on 26 September 2021 Av. Prof. Lineu Prestes 580, 05508-000 Saõ Paulo, Saõ Paulo, Brazil

MS 06-070: Received 9 February 2006/Accepted 3 August 2006

ABSTRACT

The viability of Lactobacillus paracasei and its effect on growth of the microbiota in potentially probiotic and synbiotic fresh cheeses during storage at 4 Ϯ 1ЊC was investigated. Three cheese-making trials (T1, T2, and T3) were prepared in quadruplicate, all supplemented with a Streptococcus thermophilus culture. L. paracasei subsp. paracasei was added to cheeses in T1 and T2, and inulin was added to cheeses in T2. Counts of L. paracasei, S. thermophilus, coliforms, Escherichia coli, Staphylococcus spp., DNase-positive Staphylococcus, and yeasts and molds were monitored during storage for up to 21 days. Viable counts of L. paracasei in probiotic (T1) and synbiotic (T2) cheeses remained above 7 log CFU/g during the entire storage period, whereas counts of S. thermophilus remained above 9.5 log CFU/g for cheeses from T1, T2, and T3. Populations of coliforms, Staphylococcus spp., and DNase-positive Staphylococcus were higher in T3 cheese and differed signiﬁcantly from those in cheeses from T1 and T2 (P Ͻ 0.05). Inhibition of contaminants prevailed when both L. paracasei and S. thermophilus were present in fresh cream cheese and probably was due to acid production by both strains; bacteriocin production was not found. Addition of inulin in T2 did not impact microbial viability (P Ͼ 0.05). L. paracasei subsp. paracasei in coculture with S. thermophilus was inhibitory against microbial contaminants in fresh cream cheese with or without the addition of inulin, indicating the potential use of this combination in a probiotic and synbiotic product.

There is increasing interest in developing dairy prod- approach of directly introducing live bacteria into the colon ucts containing lactic acid bacteria (LAB) as biopreserva- through dietary supplementation, another approach to in- tives. These bacteria inhibit the growth of other microor- creasing the number of beneficial bacteria such as bifido- ganisms through the activity of several compounds, includ- bacteria in the intestinal microbiota is through the use of ing organic acids, hydrogen peroxide, alcoholic com- prebiotics. Prebiotics are nondigestible dietary components pounds, diacetyl, and bacteriocins. The inhibitory activity that pass into the colon and selectively stimulate the proof LAB creates a hostile environment for pathogens and liferation and/or activity of populations of desirable bacteria spoilage organisms in food (40). Lactobacillus species are in situ. Because of potential synergy between probiotics and frequently responsible for antagonistic activity against food prebiotics, foods containing a combination of these ingre- spoilage and pathogenic microorganisms (46). LAB of the dients are often referred to as synbiotics (33). Fructooli- Lactobacillus casei group are facultatively heterofermen- gosaccharides and inulin-type fructans have been among tative; this group includes phenotypically and genetically those compounds most studied as prebiotics (18, 20, 32). heterogeneous strains of L. casei, Lactobacillus paracasei, Inulin-type fructans are known for their bifidogenic ef- Lactobacillus zeae, and Lactobacillus rhamnosus (16, 45). fect, i.e., their ability to selectively increase the number of These strains have been widely studied with respect to their bifidobacteria in the human colon; bifidobacteria are able health-promoting properties and are currently employed as to use inulin-type fructans as a sole energy source (18, 30, probiotic or mixed cultures in the food industry (24, 45). 38). Growth of colonic microbiota (other than bifidobacter- Probiotics have been defined as live microbial food ia) using inulin-type fructans as an energy source has been supplements that benefit the health of consumers by main- reported for various bacteria (14), including strains of Lac- taining or improving intestinal microbial balance (19, 33). tobacillus acidophilus and L. paracasei (25, 30). Kaplan However, this definition has evolved (43), and the most and Hutkins (25) verified that 12 of 16 Lactobacillus strains recent definition states that probiotics are live microorgan- fermented fructans with 2 to 4 degrees of polymerization isms administered in adequate amounts to positively affect (fructooligosaccharides). Makras et al. (30) investigated 10 the health of the host (17, 43). In addition to the probiotic strains of lactobacilli for their capacity to degrade oligo- * Author for correspondence. Tel: ϩ55-11-30912378; Fax: ϩ55-11- fructose and inulin-type fructans and verified that only two 38156386; E-mail: [email protected]. strains, L. acidophilus IBB 801 and L. paracasei subsp. J. Food Prot., Vol. 70, No. 1 BIOPRESERVATION OF FRESH CREAM CHEESE WITH L. PARACASEI 229 paracasei 8700:2, were capable of degrading oligofructose, achieve 9 log CFU/liter (probiotic culture). Calcium chloride (0.25 whereas only the human isolate L. paracasei subsp. para- g/liter) also was added in all trials. In the next step, all vats were casei 8700:2 degraded the long-chain inulin, growing rap- held at 42ЊC until the pH reached 6.3 to 6.4. At this time, com- idly when using both oligofructose and inulin as energy mercial rennet Ha-la (50 mg/liter) was added to the cheese-milk, sources. Few researchers have evaluated the growth of LAB which was then held until the curd formed. The curd was gently cut into cubes, placed in sterilized cotton cheesecloth, and allowed other than bifidobacteria in dairy products such as yogurts to drain at 15ЊC for 6 h. Part of the whey removed during draining and cheeses that contain inulin-type fructans. of the cheese base for production of fresh cream cheese in T2 was Fresh cream cheese is a cheese obtained from the ho- placed in sterilized screw-cap flasks, immediately refrigerated at mogenization of a fresh cheese base with addition of other 4 Ϯ 1ЊC, and subsequently used to dissolve the inulin. After drain- ingredients, including gums, hydrocolloids, salt, and spices, ing, the cheese base was cut, placed in sterilized beakers covered and possible addition of additives such as inulin-type fruc- with a polyvinyl chloride film, and incubated at 13ЊC overnight tans. Similar to other cream cheeses, fresh cream cheese is to acidify to pH values of 5 or less. On the following day, the used as a spread on bread and in sandwiches and as a salad beakers containing the cheese base were stored at 4 Ϯ 1ЊC until Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/1/228/1680018/0362-028x-70_1_228.pdf by guest on 26 September 2021 dressing. Because of its manufacturing process, fresh homogenization after the addition of the rest of the ingredients. cheese (including fresh cream cheese) appears to be an ideal Cheese base proportions of 98.7 and 74.7% were used to produce carrier for probiotic bacteria. Fresh cheese is unripened; the cheeses in T1 and T3 and the cheese in T2, respectively. The cheeses in all three trials (T1, T2, and T3) were prepared by add- thus, storage occurs at refrigeration temperatures, shelf life ing NaCl (0.8% of final product) and xanthan gum (0.5% of final is limited, and no prolonged periods of ripening are nec- product) to the fresh cream cheese. For cheese in T2, one part of essary (21). However, fresh cheese, especially fresh cream inulin Raftiline HP-Gel, which had been dissolved in two parts of cheese, provides a perfect environment for the survival and whey and heated at 55 to 60ЊC to achieve 24% of the total ingre- growth of spoilage and pathogenic microorganisms in a dients in this trial, was immediately incorporated into the cheese short period of time because of high moisture, low salt con- base by homogenization, and a smooth and homogeneous cream centration, and the absence of preservatives. was formed. The aim of the present study was to investigate the After homogenization, cheeses were packaged in individual effect of L. paracasei subsp. paracasei culture on microbial plastic cups, each containing 40 g of cheese, sealed with a metallic Ϯ Њ contaminants. A potentially probiotic L. paracasei subsp. cover, and stored at 4 1 C for up to 21 days. The following paracasei strain was added in coculture with Streptococcus day, samples of the final product were taken for microbiological and physicochemical analysis. thermophilus during manufacture of fresh cream cheese with and without the addition of the prebiotic inulin, and Sample collection. Fresh cream cheeses from each trial were the cheese product was monitored during storage at 4 Ϯ analyzed during manufacturing (day 0), on day 1 (final product), 1ЊC for up to 21 days. and after 7, 14, and 21 days of storage. On each sampling day, at least six cups containing fresh cream cheese from the same batch MATERIALS AND METHODS and trial were used for analysis. For microbiological analysis, the fresh cream cheese from two cups was thoroughly mixed with a Ingredients used for manufacture of fresh cream cheeses. sterile spoon and 25-g portions were collected aseptically from The following commercial ingredients were used to produce fresh these two cups. Portions of fresh cream cheese from the other cream cheeses: high temperature–short time pasteurized milk cups also were collected for physicochemical analysis. (Xandoˆ, Fazenda Colorado, Araras, Brazil), a starter culture of S. thermophilus TA 040 (Danisco, Dange´, France), a potentially pro- Physicochemical analysis of fresh cream cheeses. The pH biotic culture of L. paracasei subsp. paracasei LBC 82 (Danisco), values of cheeses were determined for triplicate samples with a calcium chloride (Synth, Labsynth, Diadema, Brazil), rennet Ha- pH meter (model 300 M, Analyser, Saõ Paulo, Brazil) equipped la (88 to 92% bovine pepsin and 8 to 12.5% bovine chymosin; with a penetration electrode (model 2AO4 GF, Analyser). Titrat- Christian Hansen, Valinhos, Brazil), prebiotic fiber inulin (Rafti- able acidity was determined for duplicate samples according to line HP-Gel, Orafti, Oreye, Belgium), NaCl (Cisne, Refinaria Na- the appropriate standard methods and was expressed as the acidity cional de Sal, Cabo Frio, Brazil), and xanthan gum (Rhodigel 80, in normal solution (milliliters per 100 g) (23). Rhodia, Melle, France). Microbiological analysis of fresh cream cheeses. Counts of Fresh cream cheese manufacture. Three pilot-scale fresh L. paracasei for probiotic cheeses from T1 and T2 and of S. ther- cream cheese–making trials (T1, T2, and T3) were performed in mophilus, coliforms, Escherichia coli, Staphylococcus spp., DN- quadruplicate; a fifth batch for each trial was produced only for ase-positive Staphylococcus, and yeasts and molds for cheeses determination of pH. S. thermophilus TA 040 was used as the from T1, T2, and T3 were monitored during manufacture (before starter culture in all trials to produce the cheese base. Cream homogenization) and storage. Numbers of coliforms, E. coli, cheeses in T1 and T2 contained the potential probiotic culture L. Staphylococcus spp., DNase-positive Staphylococcus, and yeasts paracasei subsp. paracasei LBC 82 and the L. paracasei subsp. and molds were also monitored in milk used for cheese manufac- paracasei probiotic strain with the prebiotic fiber inulin, respec- ture. Populations of DNase-positive Staphylococcus were moni- tively. Cream cheeses in T3 (control fresh cream cheeses) were tored in only two batches (replicates) from T1 and T3 and in only supplemented with only S. thermophilus. one batch from T2. For microbiological analysis, 25-g portions of The cheese base was manufactured in 10-liter vats from com- duplicate cheese samples were blended with 225 ml of 0.1% pep- mercial pasteurized milk heated to 42 to 43ЊC; cultures were add- tone water in a Bag Mixer 400 (Interscience, St. Nom, France) ed at that temperature. Both cultures were freeze-dried commercial and serially diluted with the same diluent. cultures for direct vat inoculation and were added at 30 mg/liter Numbers of L. paracasei were determined by pour plating 1 to achieve 10 log CFU/liter (starter culture) and at 10 mg/liter to ml of each dilution in deMan Rogosa Sharpe (MRS) agar (Oxoid 230 BURITI ET AL. J. Food Prot., Vol. 70, No. 1

TABLE 1. Changes in pH and titratable acidity of fresh cream cheeses at the time of manufacture (day 0) and during storage at 4 Ϯ 1ЊCa pH Titratable acidity (ml/100 g) Storage day T1 T2 T3 T1 T2 T3

0 5.11 Ϯ 0.22 A a 5.00 Ϯ 0.13 B a 5.15 Ϯ 0.24 A a 1 5.05 Ϯ 0.21 A b 4.85 Ϯ 0.08 B b 5.01 Ϯ 0.28 A b 0.91 Ϯ 0.16 A a 0.81 Ϯ 0.09 A a 0.89 Ϯ 0.21 A a 7 4.91 Ϯ 0.23 A c 4.75 Ϯ 0.07 B c 4.90 Ϯ 0.25 A c 1.19 Ϯ 0.19 A b 1.07 Ϯ 0.09 A b 1.17 Ϯ 0.26 A b 14 4.81 Ϯ 0.15 A d 4.63 Ϯ 0.06 B d 4.80 Ϯ 0.23 A d 1.27 Ϯ 0.19 A c 1.12 Ϯ 0.08 A c 1.23 Ϯ 0.25 A c 21 4.80 Ϯ 0.15 A d 4.69 Ϯ 0.05 B d 4.83 Ϯ 0.18 A d 1.32 Ϯ 0.19 A d 1.16 Ϯ 0.07 A d 1.27 Ϯ 0.27 A d a Values are mean Ϯ standard deviation of ﬁve batches (repetitions) of each cheese for pH and four batches for titratable acidity. T1, L. paracasei; T2, L. paracasei plus inulin; T3, control. Within each parameter (pH and acidity) and each row, means with different

small cap letters are signiﬁcantly different (P Ͻ 0.05). Within each column, means with different lowercase letters are signiﬁcantly Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/1/228/1680018/0362-028x-70_1_228.pdf by guest on 26 September 2021 different (P Ͻ 0.05).

Ltd., Basingstoke, UK) acidified to pH 5.4 with acetic acid after RESULTS AND DISCUSSION 3 days of anaerobic incubation (Anaerobic System Anaerogen, Oxoid) at 37ЊC (35). S. thermophilus was quantified by pour plat- Physicochemical analysis of fresh cream cheeses. ing 1 ml of each dilution in M17 agar with added lactose (Oxoid) Changes in pH and titratable acidity during storage are followed by incubation at 37ЊC (35, 37) for 48 h. Populations of shown in Table 1. In cheeses from T1, T2, and T3, pH Staphylococcus spp. were determined by surface plating 0.1 ml of decreased and titratable acidity increased between the day each dilution on Baird-Parker agar (with egg yolk–tellurite emul- of production (day 0) and day 21 of storage. The reduction sion; SR054C supplement, Oxoid) followed by incubation at 37ЊC in pH was significant between days 0 and 1 and after 7 and for 48 h. Coliforms and E. coli, DNase-positive Staphylococcus, 14 days of storage (P Ͻ 0.05) for the three cheeses. Ti- and yeasts and molds were quantified by plating 1 ml of each tratable acidity increased significantly after every week of dilution on Petrifilm EC Count Plates (3M Microbiology, St. Paul, storage for cheeses from T1, T2, and T3 (P Ͻ 0.05). The Minn.), Petrifilm Staph Express Count Plates (3M Microbiology), cheese from T2 had a significantly lower pH (P Ͻ 0.05) and Petrifilm YM Count Plates (3M Microbiology), respectively, than did the cheeses from T1 and T3 because of initial followed by incubation at 37ЊC for 24 h (Petrifilm EC and Petri- lower pH values in the fresh cheese base. However, titrat- film Staph Express) or 25ЊC (Petrifilm YM) for 5 days. able acidity in cheeses from T1, T2, and T3 was not sig- Evaluation of inhibitory activity of L. paracasei. L. para- nificantly different during storage (P Ͻ 0.05). casei subsp. paracasei LBC 82 was tested for inhibitory activity The reduction in pH and increase in acidity observed against two strains of Lactobacillus sakei (1 and ATCC 15521), during storage is a natural process caused by the continuous one strain of Listeria monocytogenes (IAL 633, serotype 1/2a, production of lactic acid and other organic acids by the S. Instituto Adolfo Lutz, Official Health Laboratory, Saõ Paulo, Bra- thermophilus starter culture in cheeses from T1, T2, and T3 zil), and one strain of Staphylococcus aureus (ATCC 29213) using and by the L. paracasei probiotic culture in cheeses from the spot-on-lawn assay on MRS agar and Trypticase soy agar (Ox- T1 and T2. In a previous study conducted by our research oid) supplemented with 0.5% yeast extract (TSAYE). The nature group with Minas fresh cheese, a typical Brazilian fresh of the inhibition was verified according to the method of Lewus cheese (8), a faster rate of acidification was observed when et al. (29). Proteases type XIV from Streptomyces griseus (Sigma) and ␣-chymotripsin type II from bovine pancreas (Sigma) were a mesophilic starter culture of Lactococcus lactis subsp. used to determine whether the inhibitor was proteinaceous. The lactis plus L. lactis subsp. cremoris was used without L. p. bacteriocin-producing strain L. sakei 1 (vacuum-packed pork sau- paracasei LBC 82 as a potential probiotic (7). sage isolate, Department of Clinical Analysis, College of Phar- Viability of L. paracasei and S. thermophilus during maceutical Sciences, Ribeiraõ Preto–University of Saõ Paulo, Saõ Paulo, Brazil) (12, 13, 31) was used as a positive control for fresh cream cheese production and storage. Table 2 bacteriocin production. shows the mean concentrations of S. thermophilus and L. paracasei during production and storage of fresh cream Experimental design and statistical analysis. The experi- cheeses from T1, T2, and T3. No significant differences in mental treatments and levels constituted a randomized complete S. thermophilus concentrations were seen for the three block design replicated four times (five times for pH), with re- cheeses (P Ͼ 0.05). Mean concentrations remained similar peated measurements taken at five time points. The treatments had during storage and always were above 9.5 log CFU/g for a factorial structure. An analysis of variance was used to deter- all three trials. Concentrations of the starter culture in- mine significant differences (P Ͻ 0.05) in pH, titratable acidity, creased significantly between day 0 and day 1 of storage and counts of L. paracasei and S. thermophilus between the different types of product and at different storage times employing and decreased significantly between day 1 and day 21 (P Ͻ a mixed-effects model (34). A binomial exact test was used to 0.05) for cheeses from T1 and T3. The same trend was determine significant differences (P Ͻ 0.05) in populations of co- observed for cheese from T2, probably influenced by dif- liforms, E. coli, Staphylococcus spp., DNase-positive Staphylo- ferences in the standard deviation for this trial on days 0, coccus, and yeasts and molds between the different products (11). 1, and 21. However, this variation observed for cheeses J. Food Prot., Vol. 70, No. 1 BIOPRESERVATION OF FRESH CREAM CHEESE WITH L. PARACASEI 231

TABLE 2. Viability of S. thermophilus and L. paracasei in fresh cream cheeses at the time of manufacture (day 0) and during storage at 4 Ϯ 1ЊCa Bacterial concn (log CFU/g) Storage Culture day T1 T2 T3

Streptococcus thermophilus 0 9.34 Ϯ 0.70 A a 9.77 Ϯ 0.14 A a 9.54 Ϯ 0.50 A a 1 9.86 Ϯ 0.23 A b 9.77 Ϯ 0.32 A b 9.92 Ϯ 0.16 A b 7 9.71 Ϯ 0.29 A bc 9.86 Ϯ 0.09 A bc 9.67 Ϯ 0.29 A bc 14 9.72 Ϯ 0.23 A bc 9.84 Ϯ 0.10 A bc 9.72 Ϯ 0.31 A bc 21 9.59 Ϯ 0.33 A ac 9.72 Ϯ 0.22 A ac 9.67 Ϯ 0.39 A ac

Lactobacillus paracasei 0 6.62 Ϯ 0.38 A a 6.83 Ϯ 0.08 A a NGb 1 7.20 Ϯ 0.17 A b 7.12 Ϯ 0.05 A b NG Ϯ Ϯ 7 7.19 0.10 A bc 7.25 0.17 A bc NG Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/1/228/1680018/0362-028x-70_1_228.pdf by guest on 26 September 2021 14 7.26 Ϯ 0.14 A bc 7.32 Ϯ 0.21 A bc NG 21 7.31 Ϯ 0.23 A c 7.39 Ϯ 0.24 A c NG a Values are mean Ϯ standard deviation. T1, L. paracasei; T2, L. paracasei plus inulin; T3, control. Within a row, means with different capital letters are significantly different (P Ͻ 0.05). Within a column for each culture, means with different lowercase letters are significantly different (P Ͻ 0.05). b NG, no significant growth (less than 2.00 log CFU/g). from T1, T2, and T3 was very small (less than 0.3 log were more inhibitory toward starter cultures (including S. CFU/g for all three cheeses). Vinderola et al. (48) studied thermophilus) than vice versa, i.e., S. thermophilus did not Argentinean fresh cheese supplemented with several pro- exert any effect on the growth of probiotic bacteria, with biotic microorganisms and observed that concentrations of some exceptions. The authors studied these interactions the S. thermophilus starter culture remained between 8.46 through growth kinetics and identified four different kinds and 9.33 log CFU/g, very similar to those observed in the of behavior between LAB starter and probiotic bacteria: present study. stimulation, delay, complete inhibition of growth, and no L. paracasei populations increased significantly by interactive effect. about 0.5 log CFU/g in cheeses from T1 and T2 between In one of only a few studies available that have focused day 0 and day 1 (P Ͻ 0.05) (Table 2). During the entire on growth of LAB in dairy products containing inulin-type storage period, the probiotic culture always remained at Ͼ7 fructans, O¨ zer et al. (36) reported no increased viability of log CFU/g, and the viability profile for this organism was S. thermophilus, Lactobacillus delbrueckii subsp. bulgari- very similar in both cheeses, with a slight but significant cus or L. acidophilus in yogurt supplemented with 0.5 and increase in concentrations between days 1 and 21 of storage 1.0% (wt/vol) inulin. However, the authors reported an in- (P Ͻ 0.05). However, this variation was very small (only crease in the viability of Bifidobacterium bifidum. Recently, 0.2 log). The production of probiotic foods that maintain Capela et al. (9) reported that the addition of 2% (wt/vol) specific probiotic strains at suitable concentrations during solutions of three commercial fibers, resistant starch of corn normal product shelf life is a technological challenge (26). Hi-maize, inulin Raftiline ST, and particularly fructooligo- Several researchers have proposed a minimum daily dose saccharide Raftilose P95, improved the viability of L. aci- of 108 to 109 CFU, which corresponds to 100 g of a food dophilus 33200, L. casei 279, Bifidobacterium longum 536, product containing 106 to 107 CFU/g (3, 22, 28). Based on and L. rhamnosus GG in fresh yogurt (compared with con- this criterion, concentrations of L. paracasei in fresh cream trol yogurt without fibers) during 4 weeks of storage at 4ЊC. cheeses from T1 and T2 remained sufficiently high to have In our study, inulin did not affect growth or viability of probiotic potential during the entire storage period. either L. paracasei or S. thermophilus in T2 cheeses. Microbial interactions, either beneficial (cooperation) or unfavorable (inhibition), usually generate changes in the Concentrations of microbial contamination indica- composition of the microbiota during production and stor- tors in milk and fresh cream cheeses during production age of fermented milk products (2). In the present study, and storage. Concentrations of the microbial indicators of fresh cream cheeses from T1 and T2 did not differ signif- contamination, i.e., coliforms, E. coli, Staphylococcus spp., icantly in regard to viability of L. paracasei during storage DNase-positive Staphylococcus, and yeasts and molds, in (P Ͼ 0.05). Populations of S. thermophilus also were not the milk uded in production and in the resulting fresh cream significantly different in cheeses from T1, T2, and T3 (P cheeses during manufacture and storage are respectively Ͼ 0.05). Therefore, these results do not provide proof of presented in Tables 3 and 4. Except for coliform concen- an interaction between L. paracasei and S. thermophilus in trations in one sample of milk used to produce cheeses in terms of growth stimulation or suppression. On the other T2 and T3, the concentrations of all remaining microor- hand, Vinderola et al. (47) observed that all L. casei and ganisms in milk were below the limits established by Bra- Bifidobacterium strains weakly inhibited the growth of all zilian regulatory standards (5). Except for DNase-positive S. thermophilus strains tested and that probiotic bacteria Staphylococcus in one 1-day-old sample of fresh cream 232 BURITI ET AL. J. Food Prot., Vol. 70, No. 1

TABLE 3. Populations of microbial contaminants detected in commercial pasteurized milk used for cheese manufacturea Bacterial concn (CFU/ml)b

Microbial contaminant T1 T2 T3

Coliforms Ͻ1 1.00 (Ͻ1–1.00) 1.00 (Ͻ1–1.00) E. coli Ͻ1 Ͻ1 Ͻ1 Staphylococcus spp. 103 (Ͻ10–103)20(Ͻ10–20) 2.29 ϫ 102 (Ͻ10–1.29 ϫ 103) DNase-positive Staphylococcus Ͻ1 Ͻ1 Ͻ1 Yeasts and molds 1.00 (Ͻ1–1.00) 1.74 (Ͻ1–3.00) 1.00 (Ͻ1–1.00) a T1, L. paracasei; T2, L. paracasei plus inulin; T3, control. b Values are the mean of four replicates of each trial, except for DNase-positive Staphylococcus, which was evaluated in two replicates from T1 and T3 and in one replicate from T2. Means were calculated from samples in which the contaminants were detected. The

range (in parentheses) was based on all samples analyzed. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/1/228/1680018/0362-028x-70_1_228.pdf by guest on 26 September 2021 cheese from T3, the concentrations of all indicators were cus spp. during storage ranged were Ͻ2 to 4.06 log always in accordance with Brazilian regulatory standards CFU/g for cheese from T1, Ͻ2 to 3.54 log CFU/g for (4) for all batches produced. These standards establish a cheese from T2, and Ͻ2 to 4.06 log CFU/g for cheese from most-probable-number limit for coliforms in milk of Ͻ1 T3. Considering all samples analyzed during the five sam- CFU/ml, complete absence of E. coli in 1 ml of pasteurized pling periods, 65% (26 of 40 samples) and 55% (22 of 40 type A milk, and maximum concentrations of 103 CFU/g samples) of fresh cream cheeses from T1 and T2, respec- for coliforms, 102 CFU/g for E. coli, 102 CFU/g for co- tively, yielded Staphylococcus spp. concentrations above agulase-positive Staphylococcus and 5 ϫ 103 CFU/g for the limit of detection for the method used (2 log CFU/g). yeasts and molds for cheeses of very high moisture (above However, for cheese from T3, up to 87.5% of the samples 55%) and with viable LAB (4, 5) such as the fresh cream (35 of 40 samples) had Staphylococcus spp. above the limit cheese produced in this study. of detection (Table 4). Coliforms were not detected in cheeses after 7 days of Statistical analysis indicated that fresh cream cheese storage (Table 4); these bacteria probably were inhibited by from T3 had significantly higher coliform, Staphylococcus the low pH. Inhibition of coliforms as a result of acid pro- spp., and DNase-positive Staphylococcus concentrations duction by LAB also was reported by Assis et al. (1) and than did the cheeses from T1 and T2 (P Ͻ 0.05). This Rocha et al. (39) in probiotic Minas fresh cheeses. In the finding indicates that L. paracasei in coculture with S. ther- present study, E. coli was not detected in any of the samples mophilus inhibited the bacterial contaminants in both the analyzed during production and storage of fresh cream probiotic and synbiotic fresh cream cheeses. However, a cheeses from T1, T2, and T3. Populations of Staphylococ- clear linear relationship was not observed for microbial in-

TABLE 4. Populations of microbial contaminants detected in fresh cream cheeses during manufacture (day 0) and storage at 4 Ϯ 1ЊC

Bacterial concn (CFU/g)b % positive samples Microbial (no. positive/total contaminant Triala 0 day 1 day 7 days 14 days 21 days no. analyzed)c

Coliforms T1 1.40 (Ͻ1–1.60) Ͻ1 Ͻ1 Ͻ1 Ͻ1 7.5 (3/40) A T2 1.00 (Ͻ1–1.00) 1.20 (Ͻ1–1.30) 1.00 (Ͻ1–1.00) Ͻ1 Ͻ1 17.5 (7/40) A T3 1.31 (Ͻ1–1.48) 1.66 (Ͻ1–1.90) 1.39 (Ͻ1–1.48) Ͻ1 Ͻ1 22.5 (9/40) B

Staphylococcus T1 2.55 (Ͻ2–3.72) 2.92 (Ͻ2–3.98) 2.96 (Ͻ2–3.93) 2.70 (Ͻ2–4.06) 3.23 (Ͻ2–3.85) 65.0 (26/40) A spp. T2 2.24 (Ͻ2–2.48) 2.68 (Ͻ2–3.52) 2.90 (Ͻ2–3.54) 2.83 (Ͻ2–3.20) 2.73 (Ͻ2–3.30) 55.0 (22/40) A T3 2.61 (Ͻ2–3.85) 3.29 (2.30–4.02) 3.11 (2.00–3.98) 3.43 (Ͻ2–4.06) 3.38 (Ͻ2–4.03) 87.5 (35/40) B

DNase-positive T1 Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ1 0.0 (0/19) A Staphylococ- T2 Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ1 0.0 (0/9) A cus T3 Ͻ1 2.18 (Ͻ1–2.26) 1.78 (Ͻ1–1.78) 1.24 (Ͻ1–1.48) Ͻ1 31.6 (6/19) B

Yeasts and T1 Ͻ1 Ͻ1 Ͻ1 1.00 (Ͻ1–1.00) 1.82 (Ͻ1–2.64) 7.5 (3/40) A molds T2 1.00 (Ͻ1–1.00) Ͻ1 1.00 (Ͻ1–1.00) 1.00 (Ͻ1–1.00) 1.38 (Ͻ1–1.90) 20.0 (8/40) A T3 Ͻ1 1.00 (Ͻ1–1.00) Ͻ1 1.60 (Ͻ1–1.60) 2.75 (Ͻ1–3.66) 15.0 (6/40) A a T1, L. paracasei; T2, L. paracasei plus inulin; T3, control. b Values are the mean of four replicates of each trial, except for DNase-positive Staphylococcus, which was evaluated in two replicates from T1 and T3 and in one replicate from T2. Means were calculated from samples in which the contaminants were detected. The range (in parentheses) was based on all samples analyzed in each period. c For each microorganism, percentages with different letters are signiﬁcantly different (P Ͻ 0.05). J. Food Prot., Vol. 70, No. 1 BIOPRESERVATION OF FRESH CREAM CHEESE WITH L. PARACASEI 233

TABLE 5. Sensitivity of L. sakei 1, L. sakei ATCC 15521, L. in T1 and T2. In preliminary studies (data not shown), L. monocytogenes IAL 633 (serotype 1/2a), and S. aureus ATCC paracasei was unable to inhibit contaminants in fresh cream 29213 to L. paracasei subsp. paracasei LBC 82 on MRS agar and cheese produced through direct acidification with lactic acid TSAYE without the addition of starter cultures (6). Thus, inhibition Inhibition zonea of contaminants prevailed when both L. paracasei and S. Indicator thermophilus were present in the fresh cream cheese. The microorganism MRS agar TSAYE combination of acids produced by these two strains might L. sakei 1 + Ϫ have resulted in increased inhibition in the fresh cream L. sakei ATCC 15521 + Ϫ cheese. The addition of inulin to the cheese in T2 did not L. monocytogenes IAL 633 +++ Ϫ impact growth or viability of these organisms in the cheese S. aureus ATCC 29213 ++ Ϫ (P Ͼ 0.05). S. thermophilus also was not inhibitory to yeasts and molds, either alone or in coculture with L. para- a Ϫ , no zone; +, 2 cm; ++, 2.5 cm; +++, 3 cm. casei. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/1/228/1680018/0362-028x-70_1_228.pdf by guest on 26 September 2021 In several studies, Lactobacillus strains, particularly those of L. casei and L. paracasei, used alone or in cocul- dicators during storage of the cheeses from T1, T2, and T3. ture with other LAB have inhibited the growth of spoilage Thus, without a clear time effect, the main interference for and pathogenic microorganisms, including coliforms (41), microbial indicators was the groups (trial effect) rather than E. coli (10), S. aureus (10, 41, 42), L. monocytogenes, Sal- the time of storage. No significant differences in concen- monella spp. (10), and Candida spp., Zygosaccharomyces trations of yeasts and molds were found for fresh cream bailii, and Penicillium sp. (44). cheeses from T1, T2, and T3 (P Ͼ 0.05) (Table 4). The results of the present study indicate that L. para- Inhibitory activity of L. paracasei. The inhibitory ac- casei subsp. paracasei LBC 82 may be used together with tivity of L. paracasei subsp. paracasei LBC 82 is shown S. thermophilus in the production of fresh cream cheese, in Table 5. Inhibition of S. aureus, L. sakei (two strains), either with or without addition of the prebiotic inulin. The and L. monocytogenes by L. paracasei was observed in resulting cheese is a potentially probiotic and synbiotic MRS agar but not in TSAYE. Inhibition could not be at- product with inhibitory properties against microbial con- tributed to bacteriocin production because the inhibition taminants. Additional studies should be conducted to fur- was not affected by proteases. Hydrogen peroxide produc- ther characterize the product, particularly with regard to tion was rejected as a reason for inhibition due to incuba- consumer acceptance. tion under anaerobic conditions (29). Thus, inhibition of the contaminants by L. paracasei in fresh cream cheeses in the ACKNOWLEDGMENTS present study was most likely due to acid production; the The authors thank Fundaçaõ de Amparo a` Pesquisa do Estado de strains tested on the spot-on-the-lawn assay were inhibited Saõ Paulo (FAPESP) (projects 02/11294-0, 02/14185-8, 03/13748-1, 04/ only in MRS agar, which contains glucose, and not in 13597-6, and 06/51011-9) and Coordenaçaõ de Aperfeiçoamento de Pes- soal de Nı´vel Superior (CAPES) for financial support and Xandoˆ, Danisco, TSAYE, which does not contain glucose (29). Rhodia, Orafti, Clariant, and 3M Microbiology for providing part of the L. paracasei subsp. paracasei belongs to the faculta- material resources used in the present study. The authors also thank Prof. tive heterofermentative lactobacilli group, whose members Dr. Ju´lia Maria Pavan Soler and Afonso Massao Yamaguchi (Centro de ferment hexose into lactic acid. They also ferment pentose Estatı´stica Aplicada, Instituto de Matema´tica e Estatı´stica, University of with an inducible phosphoketolase to produce lactic and Saõ Paulo) for the statistical analysis and Prof. Dr. Elaine Cristina Pereira De Martinis and Carlos Eduardo Mendes D’Angelis (Departamento de acetic acids (45). In contrast, S. thermophilus is homofer- Ana´lises Clıńicas, Faculdade de Cieˆncias Farmaceûticas de Ribeiraõ Preto, mentative and converts only lactose into lactic acid (15). University of Saõ Paulo) for evaluation of the inhibitory activity of L. Production of other acids, including orotic, citric, pyruvic, paracasei. and uric acids, has been described in milk fermented with REFERENCES S. thermophilus, L. delbrueckii subsp. bulgaricus, and L. paracasei subsp. paracasei (27). 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