International Dairy Journal 19 (2009) 696–702
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Technological properties of candidate probiotic Lactobacillus plantarum strains Ralitsa Georgieva a, Ilia Iliev b, Thomas Haertle´ c, Jean-Marc Chobert c, Iskra Ivanova d, Svetla Danova a, * a
Department of Microbial Genetics, Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bontchev Str, 1113 Sofia, Bulgaria Department of Biochemistry & Microbiology, Plovdiv University, Tzar Assen Str, 4000 Plovdiv, Bulgaria c UR 1268 Biopolyme`res Interactions Assemblages, INRA, Equipe Fonctions et Interactions des Prote´ines Laitie`res, Rue de la Ge´raudie`re, B. P. 71627, 44316 Nantes Cedex 3, France d Department of General and Applied Microbiology, Biological Faculty, Sofia University, 8, Dragan Tzankov Str, 1113 Sofia, Bulgaria b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 March 2009 Received in revised form 2 June 2009 Accepted 16 June 2009
The study of new probiotic strains for their technological relevance and use in dairy products is important for trade and industry. Eight Lactobacillus plantarum strains isolated from Bulgarian cheeses and selected for their potential probiotic properties were characterized. In vitro tests with the API ZYM system revealed high aminopeptidase and phosphatase activity, and weak lipolytic activity. The L. plantarum strains showed also a weak proteolytic activity and were characterized as slow variants on the base of their coagulation ability. They maintained high viability in fermented milk over extended shelf-times at refrigerated temperature and demonstrated a good adaptation to 6% NaCl. Among the preservatives, only calcium propionate did not affect the growth of L. plantarum. The highest concentrations used of potassium sorbate (0.5 and 1%) and nisaplin (0.02%) decreased the bacterial growth. One L. plantarum strain was tested as an adjunct to commercially available formula for cream cheese. This candidate probiotic culture withstood the technological processing and retained high number of 107 cfu g1 at the end of the 3 months storage period at 4 C. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Fermented dairy products are widely accepted healthy products and valued components of food diets. The incorporation of probiotic bacteria as adjuncts in different fermented milk products is currently an important topic with industrial and commercial consequences. The food application of probiotics has reinforced the acclaimed healthy properties and given rise to an increased consumption of these products in Europe and USA (Kristo, Biliaderis, & Tzanetakis, 2003). A number of dairy products are marketed as containing probiotic bacteria. Fermented milk and cheeses have been described as the most suitable carriers for the administration of such bacteria (Lourens-Hattingh & Viljoen, 2001; Saarela, Mogensen, Fonden, Matto, & Mattila-Sandholm, 2000). A variety of microorganisms, typically food grade lactic acid bacteria (LAB), have been evaluated for their probiotic potential and are applied as adjunct cultures in various types of food products or in therapeutic preparations (Rodgers, 2008). To exert healthpromoting effects these bacteria must survive during the shelf life of the food used as a vehicle and during the passage into the gastrointestinal tract (GIT) (Kailasapathy & Rybka, 1997). However, the probiotic bacteria often show poor viability in market
* Corresponding author. Tel.: þ359 2 9793119; fax: þ359 2 8700109. E-mail address:
[email protected] (S. Danova). 0958-6946/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2009.06.006
preparations at the moment of consumption (Al-Otaibi, 2008; Gueimonde et al., 2004). Several factors can affect bacterial viability in milk products, such as conditions of fermentation, storage temperature, preservation methods and individual strain properties (Kailasapathy & Rybka, 1997; Shah, 2000). Considerable variation occurs among the strains with regard to their behaviour under industrial conditions and their overall effect on the product quality. Therefore, a proper evaluation of each candidate probiotic strain becomes an important step for further implementation as adjuncts. These cultures should not have adverse effects on the taste or aroma of the product and should not enhance acidification during the shelf life of the product (Heller, 2001). Among industrially used probiotics, Lactobacillus plantarum strains are often incorporated in different health products (De Vries, Vaughan, Kleerebezema, & de Vos, 2006). L. plantarum is one of the non-starter LAB species most frequently isolated from ripened cheeses (Rantsiou, Urso, Dolci, Comi, & Cocolin, 2008; Van Hoorde, Verstraete, Vandammea, & Huys, 2008), including the most popular traditional white cheese in Bulgaria (Chomakov & Kirov, 1973; Georgieva et al., 2008). However, according to the Bulgarian State Standard (1989) for dairy products, at present there is no practice for addition of L. plantarum strains as adjunct cultures in the commercial cheese production. In our recent study a total of 21 L. plantarum strains have been isolated from artisanal samples of Bulgarian white brined cheese (Georgieva et al., 2008). Determination of antimicrobial activity,
R. Georgieva et al. / International Dairy Journal 19 (2009) 696–702
antibiotic susceptibility and transit tolerance showed the presence of isolates with beneficial probiotic activities. Eight of these strains, selected on the base of commonly accepted in vitro criteria were used in the present study for assessment of some technologically relevant properties. With this aim, a set of experiments was performed for characterization of (i) their acidification, enzymatic and proteolytic activities, (ii) their stability in fermented milk during refrigerated storage and (iii) the effect on them of some commonly used preservatives in dairy industry. One selected strain was tested as adjunct culture in production of commercial cream cheese. 2. Materials and methods 2.1. Strains and growth conditions Eight previously selected L. plantarum strains (RL19, 22, 23, 29, 34, 36, 37, 38; Georgieva et al., 2008) were studied. They were isolated from artisanal Bulgarian white cheeses after two months of ripening. Freeze-dried cultures stored at 20 C over 1 year were used for the experiments. Before the assays, L. plantarum strains were cultured twice in de Man, Rogose Sharpe broth (MRS, Sharlau, Barcelona, Spain) at 37 C for 24 h, under anaerobic condition (BBLÒ Gas Pak Anaerobic System Envelopes, Becton Dickinson, Sparks, MD, USA). 2.2. Enzymatic activities The enzymatic activities of L. plantarum strains were assayed by the API ZYM galleries (BioMe´rieux, Lyon, France), following the manufacturer’s instructions. Cells of 24 h cultures on modified MRS agar plates, containing 0.5% (w/v) glucose were collected. After washing, obtained dense suspensions in sterile water were used in the assay. The results were graded from 0 to 5 (API ZYM units) by comparison of the observed colour with the colour reaction chart. A value of 0 corresponded to a negative reaction and 5 to a reaction of maximal intensity. 2.3. Milk coagulation and proteolytic activity The milk coagulation test was performed in 10% (w/v) reconstituted skim milk (RSM, Sharlau) and in skim milk supplemented with: 0.25% yeast extract (RSM-YE; Difco, Sparks, MD, USA), 0.25% casein enzymatic hydrolysate (RSM-CH; Fluka, Buchs, Switzerland), 0.25% peptone (RSM-P; Difco) and 1% glucose (RSM-G) at 37 and 42 C. The strains able to coagulate milk within 16 h were defined as fast coagulating strains, while slow variants require a longer period of time (more than 36 h) (He´bert, Raya, Tailliez, & de Giori, 2000). The proteolytic activity (o-phthaldialdehyde spectrophotometric assay, OPA test) was determined according to Church, Swaisgood, Porter, and Catignani (1983) by inoculation (5%, v/v) of Lactobacillus cultures, washed twice with PBS (phosphate buffered saline), in sterile reconstituted skim milk (10%, w/v) followed by incubation for 24 h at 37 C. The absorbance of the OPA reagent with aliquot of the control (10% skim milk) was subtracted from each reading. The results were expressed in L-Gly equivalent (mM L1). 2.4. Viability of L. plantarum strains in fermented milk and pH changes during cold storage Viability in milk during storage at 4 C was analysed in heattreated (110 C for 10 min) fat-free skim milk (Sharlau). Aliquots (100 mL) were inoculated with w106 cfu mL1 of each strain, previously pre-cultured in milk. After 20 h cultivation at 37 C, the fermented milk was transferred at 4 C and stored for 2 months.
697
The number of viable bacteria was determined by plate count on MRS agar (pH 5.4) at different time points: after milk inoculation (initial counts), at the end of fermentation (1 day) and after 7, 14, 21, 28 and 60 days of storage. In parallel, the acidifying ability of L. plantarum strains was determined by measurement of pH (SensoDirect pH110, LovibondÒ, Dortmund, Germany). 2.5. Influence of food preservatives on the growth of L. plantarum strains Different food preservatives were used in recommended and higher concentrations according to Codex General Standard for cheese (Codex, 2008): sodium chloride (Sigma, St. Louis, MO, USA) at 3, 6, 8, and 10% (w/v); E 202 potassium sorbate (Barentz Ingredient BV, Almelo, The Netherlands) at 0.1, 0.2, 0.5, and 1.0% (w/v); E 282 calcium propionate (Barentz Ingredient BV) at 0.3 and 0.6% (w/v) and E 234 Nisaplin (Aplin and Barrett, Ltd., Trowbridge, U.K.) at 0.01 and 0.02% (w/v). Their effect on strain growth was assessed in MRS broth, supplemented with the above cited preservatives. The analyses were performed in 96-well microplates (Nunc, Rochester, NY, USA) in a volume of 200 mL inoculated with 10% (v/v) of exponential cultures. Changes in optical density were measured in triplicate at 590 nm, after plate shaking (Organon Teknika Reader 530TC, Authos Labec. Instruments, Salzburg, Austria). The relative growth of each strain (after 24 h incubation at 37 C) was expressed as the percentage of the optical density of control culture (culture in MRS without preservative). 2.6. Viability of L. plantarum RL34 in cream cheese during storage and effect of potassium sorbate The cream cheese based on dairy ingredients (skim curd, cream with 35% fat, milk proteins, salt, hydrocolloids) was made in industrial conditions in two forms: a non-preserved control cheese (cheese without any preservative) and a cheese with 0.2% (w/w) potassium sorbate. According to the cultured cream production method (by technological instructions of Sibio-93 Ltd, Plovdiv, Bulgaria) different steps were followed: standardization, pasteurization (30 min at 80–82 C) and homogenization of the cream, addition of dry ingredients under high shear and heat treatment, fermentation, homogenization and filling. The fermentation process started with an addition of 1% (v/v) inoculum of L. plantarum RL34, previously activated in 10% skim milk (EPI Ingredients, Ancenis, France) for 12 h and containing a minimum of 2–4 106 cfu mL1 bacteria. After 4 h fermentation process at 37 C, the cream cheese samples were aseptically filled in 100 g market plastic boxes, rapidly cooled, and stored at 4 C during commercially recommended period. Counting of L. plantarum cells (cfu g1) was performed at the end of manufacturing process (0 point) and at different time intervals during 2 months of storage (1, 7, 14, 21, 30, 40, 60 days). Samples (10 g) of both variants of cream cheese aseptically suspended in 90 mL 2% (w/v) sodium citrate solution and homogenized (Stomacher Lab Blender 400, Humeau Laboratory, La Chapelle-sur-Erdre, France) were analysed by standard dilution method on MRS agar (pH 5.4) plates. All experiments were performed in triplicate. The yeast contamination was monitored on Sabouraud agar plates for both variants of the cheese and for commercial samples without LAB addition. In parallel, the pH change in the products was determined. 2.7. Statistical analysis Statistical evaluation of the changes between the groups was carried out using two tailed Student’s t-test and GraphPad Prism
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4.0. A p-value below 0.05 was considered to be statistically significant. 3. Results and discussion In the present study eight L. plantarum strains determined as putative probiotics were investigated for their technologically relevant properties. In vitro trials were performed with cultures obtained by freeze-drying and subsequent long-term storage at low temperature. All L. plantarum strains showed good survival under these conditions (unpublished data), which could be important for their use into food systems. 3.1. Enzymatic activity The characterization of the enzyme activity is of major importance for strain selection as adjunct cultures and for prediction of their physiological activity and influence on the final quality of the product (Arora, Lee, & Lamoureux, 1990). For this purpose, the enzymatic potential of eight L. plantarum strains was assessed by the API ZYM system on semi-quantitative scale from 0 to 5. The strains showed maximum peptidase activities of 5 (leucine- valineand cystine-aminopeptidase) (Fig. 1), but no detectable proteinase activities (trypsin and chymotrypsin). Phosphatase (acid and alkaline) and phospho-hydrolase activities in the range of 3–5, moderate esterase-lipases (C8) (mean activities of 2.5) and relatively weak esterase (C4) and lipase (C14) activities were detected. b-Galactosidase, a- and b-glucosidase and N-acetyl-b-glucosaminidase activities were high (in the range of 4–5) in all strains. The only exception was a-glucosidase for RL19 which activity was 0.5 API ZYM units. Low a-galactosidase activity (0.5 unit) and no amannosidase and a-fucosidase activity were observed for all tested cultures. Overall, tested L. plantarum strains showed similar enzyme profiles, reported earlier for this species (Kostinek et al., 2007; Papamanoli, Tzanetakis, Litopoulou-Tzanetaki, & Kotzekidou, 2003; Pulido et al., 2007). Higher cystine-aminopeptidase activity for studied cheese strains was detected in comparison with lactobacilli isolated from other sources (Herreros, Fresno, Gonza´lez Prieto, & Tornadijo, 2003; Papamanoli et al., 2003). The observed activities are in agreement with the existence of different genes encoding intracellular peptidases in the genome of L. plantarum (Kleerebezem et al., 2003). The presence of lactobacilli with high aminopeptidase activity is advantageous for cheese ripening and flavour development. Although Pulido et al. (2007) describe good lipolytic activity for some obligatory heterofermentative lactobacilli, commonly they are weakly lipolytic. Indeed, low levels of lipolysis are recommended for cheese production as LAB are present usually at high numbers during the ripening period and thus, are responsible for the liberation of significant levels of free fatty acids. High level of lipolysis results in a bitter taste and flavour of the cheese product (Collins, Mc Sweeney, & Wilkinson, 2003). The high acid phosphatase and acid phospho-hydrolase activities of tested L. plantarum strains may be useful in metabolizing phosphates in the acidic external environment prevalent in cheese maturation (Arora et al., 1990). Molecular analyses of the genome diversity predicted the capacity of this versatile species to metabolise different carbon sources (Molenaar et al., 2005) and the range of fermented sugars probably related to the habitat of each strain. The high galactosidase and glucosidase activities detected, and relatively low activities toward other carbon sources, suggest that L. plantarum strains prefer glucose and lactose for their carbon and energy requirement. In addition, the presence of these enzymes may be beneficial for the utilization
of galacto- and gluco-oligosaccharides commonly used lately as prebiotic ingredients in dairy industry. 3.2. Milk coagulation and proteolytic activity The L. plantarum strains were further analysed for their ability to coagulate skim milk and milk supplemented with additional nitrogen or carbon sources. All strains did not coagulate milk within 16 h and were characterized as slow variants (Table 1). Nevertheless, most of them coagulated milk supplemented with yeast extract, casein enzymatic hydrolysate and peptone, but not milk supplemented with glucose. Only the strain RL19 did not show any coagulation activity. These results were obtained at optimal temperature (37 C), while at technologically relevant temperature (42 C) none of the strains had coagulation activity within the experimental time scale, even after addition of nitrogen source. The proteolytic activity varied from 0.170 to 0.609 mM Gly L1 into the group of L. plantarum strains (Table 1). The highest activity was detected for strain RL34. The measured low proteolysis correlated with the negligible capacity of the L. plantarum strains to hydrolyse milk proteins (as1-, as2-, b-, k-caseins, b-lactoglobulin and a-lactalbumin) estimated by SDS-PAGE (unpublished data). Probably the protein degrading machinery of L. plantarum is limited in enzymes for large-polypeptide utilization. In silico genome analysis of L. plantarum WCFS1 showed the absence of genes encoding extracellular proteases (Kleerebezem et al., 2003). Low proteolytic activity and variable coagulation ability usually were reported for cheese originated L. plantarum strains in comparison with other LAB assayed in the same conditions (Briggiler-Marco et al., 2007; Herreros et al., 2003). However, this is not a drawback for their use as adjunct cultures in the cheesemaking process (Crow, Curry, & Hayes, 2001). Lactobacillus strains with low proteolytic activity are successfully applied as adjunct cultures in different types of cheeses. They contribute in the secondary proteolysis via their peptidolytic potential, increasing the amount of small peptides and free amino acids, major precursors of specific flavour compounds (Briggiler-Marco et al., 2007; Lynch, Muir, Bancs, Mc Sweeney, & Fox, 1999). 3.3. Viability of L. plantarum strains in fermented milk and pH changes during cold storage The test for viability and change of pH was determined in sterile skim milk during the cold storage for two months. The exponential L. plantarum cultures were inoculated into the milk at the beginning of the fermentation process and the average initial microbial counts were 4.0 1.8 106 cfu mL1. All strains showed significant growth during fermentation with 2 log increase in the cell number. During the cold storage until day 28 a good viability ranging from 6.8 to 7.5 log orders, for different L. plantarum strains was detected (Fig. 2). Only after two months of storage a significant reduction (p < 0.001) was observed, with average viable cell levels of 4.5 1.9 105 cfu mL1. For tested L. plantarum strains a lower reduction in viability (0.8–1.5 log orders) was detected, in comparison with other probiotic strains of Lactobacillus acidophilus after four weeks storage (Damin, Minowa, Alcantara, & Oliveira, 2006; Vinderola, Bailo, & Reinheimer, 2000). Although there are no set standards concerning the population of the probiotic organism at the end of the product shelf life, the minimum levels of 106 cfu mL1 is usually considered as acceptable to provide their benefits (Samona & Robinson, 1994). In this aspect the obtained results indicate the fermented milk as a suitable carrier for L. plantarum strains and predict good viability with high numbers of living cells in the final product at the moment of consumption. Because of the possible reduction of the probiotic organisms during
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699
Esterase
Esterase-lipase
Lipase
Leucine arylamidase
Valine arylamidase
Cystine arylamidase
Enzymes
Trypsin
Acid phosphatase
Alkaline phosphatase
Phosphohydrolase
α-Galactosidase β-Galactosidase β-Glucuronidase α-Glucosidase β-Glucosidase
N-acetyl-β-glucosaminidase 0
1
2
3
4
5
Activity Fig. 1. Determination of enzyme activities of L. plantarum strains in the scale from 0 (no activity) to 5 (maximal activity) by API ZYM galleries. L. plantarum strains were: ( ), RL38; ( ), RL37; ( ), RL36; (,), RL34; ( ), RL29; (E), RL23; ( ), RL22, (-), RL19. Chymotrypsin, a-mannosidase and a-fucosidase activities were not detected in any strain.
Table 1 Test for milk coagulation and proteolytic activity of L. plantarum strains. Strain
RL19 RL22 RL23 RL29 RL34 RL36 RL37 RL38
Milk coagulationa
Proteolytic activityb (mM Gly L1)
RSM
RSM-CH
RSM-YE
RSM-P
RSM-G
þ þ þ þ þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ
0.217 0.277 0.170 0.348 0.609 0.541 0.289 0.571
0.041 0.044 0.031 0.038 0.095 0.048 0.076 0.043
a Strains were inoculated (2% v/v) into: reconstituted skim milk (RSM), RSM supplemented with casein enzymatic hydrolysate (RSM-CH), RSM supplemented with yeast extract (RSM-YE), RSM supplemented with peptone (RSM-P), or RSM supplemented with glucose (RSM-G), and incubated for 16 h at 37 C. Values are means of four independent trials; þ denotes milk coagulation. b Values are means (SD) of three replicate evaluations at 24 h for each bacterial strain.
R. Georgieva et al. / International Dairy Journal 19 (2009) 696–702
** * **
***
***
5 4 0
1
7
14
21
28
8
6.0
7 6
9
6.5
5.5 5.0
7.0 6.5 *
7
5.5 ** **
6
4.5
5
4.0
4
5.0 4.5 4.0 0
60
1
8
6.5 6.0
* **
5.5 6
*** ***
5
5.0 4.5 4.0
4 1
7
14
14
21
28
60
21
28
7.0
9
60
Time (day)
L.plantarum (log cfu mL-1)
7.0
pH
L.plantarum (log cfu mL-1)
9
0
7
Time (day)
Time (day)
7
6.0
*
pH
***
7.0
6.5
8
* 6.0
7
5.5 *
6
***
5
pH
**
8
**
L.plantarum (log cfu mL-1)
L.plantarum (log cfu mL-1)
9
pH
700
5.0 4.5 4.0
4 0
1
7
14
21
28
60
Time (day)
Fig. 2. Viability of L. plantarum strains ( , ) and pH change (;; A) of fermented milk during cold storage. Values are means (SD) of three replicate evaluations for each bacterial strain; t-test: *p < 0.05; **p < 0.01; ***p < 0.001.
passage through the GIT, a therapeutic daily dose recommended is 108–109 cfu g1 (Kailasapathy & Rybka, 1997, Shah, 2000). The level is not constant and it may vary according to the species and the strain used. Thus, to estimate the needed daily intake of the potentially probiotic strains, in vivo assay is necessary. The initial pH value of the milk (6.4) decreased and ranged from 4.6 to 4.9 at the end of fermentation (Fig. 2). During the cold storage, a detectable drop in pH occurred between day 14 and day 21 for six of the studied strains. Low acidifying ability and no significant pH change was detected for strain RL19 and RL29 (with a final pH of 5.3 and 5.1, respectively) (Fig. 2). For the other L. plantarum strains pH values ranged near 4.5 0.1 (up to 28 days) and corresponded to the desired pH values recommended for such fermented products (Ro¨nka¨ et al., 2003). 3.4. Influence of food preservatives on growth of L. plantarum strains Use of preservatives is one of the factors affecting strain survival in the final product. Many different preservatives with antimicrobial effect are added in the food systems to extend the shelf life of the foods. They prevent the development of undesirable microflora, but also can affect the starter or probiotic bacteria used in the manufacturing process (Gomes, Teixeira, & Malcata, 1998). The effect of widely used dairy preservatives on the growth of L. plantarum strains was evaluated (Table 2). Among the tested chemical preservatives, only calcium propionate, at both concentrations studied, did not interfere with the growth of L. plantarum cultures and it even had a slight stimulatory effect. In the presence of 3% NaCl and 0.1% (w/v) potassium sorbate all strains showed significant growth. A good adaptation was observed in the presence of 6% NaCl (w/v) with a growth of about 60% of that of the control, while no growth was measured in the presence of 10% NaCl (w/v). Potassium sorbate in concentrations of 0.5 and 1% (w/v) inhibited the growth of L. plantarum strains in different degrees (15–60% and 54–74%, respectively; Table 2). A significant growth (80%) was
measured for seven of the studied strains in the presence of 0.01% (w/v) nisaplin. However, 0.02% (w/v) nisaplin was strongly inhibitory for most of them. NaCl is an essential ingredient used in food industry to boost the sensory characteristics and to satisfy the human daily requirement. Additionally, NaCl is widely used as a preservative in the production of long storage cheeses and is important for controlling cheese ripening (Guinee & Fox, 2004). Depending on the type of cheese, its content varies from 0.1 to 4.2% (Food Standards Agency, 2002). In our work, L. plantarum strains appeared to be sensitive only to the highest tested concentrations of NaCl. Thus the salt concentrations generally used in cheese-making processes, will not affect their viability. In comparison, L. plantarum strains isolated from dry fermented sausage were able to grow in the presence of 6.5–10% NaCl (w/v) (Papamanoli et al., 2003). In the study of processes requiring high salt concentrations Rao, Pintado, Stevens, and Guyot (2004) determined that 4% NaCl and a pH of 6.0–6.6 are the most suitable conditions for starter application of two L. plantarum strains. Potassium sorbate and calcium propionate are generally recognized as safe (GRAS) food additives and are recommended as effective chemical preservatives for many types of cheeses with a final pH of 6.0–6.5 or less. They have a strong antimicrobial effect against different yeasts, moulds and bacteria (Stopforth, Sofos, & Busta, 2005). Results in Table 2 show sufficient growth for all tested L. plantarum strains in the presence of preservatives at the concentrations used in food production. Therefore, they could be applied as adjuncts cultures in cheeses protected with these preservatives. Nisin is a broad-spectrum bacteriocin permitted in the USA and many countries in Europe for the preservation of processed cheeses and other dairy products under the trade name Nisaplin (Delves-Broughton, Blackburn, Evans, & Hugenholtz, 1996). The activity of nisin against starter and probiotic bacteria varies (Kot, Murad, & Bezkorovainy, 2001; Vinderola, Costa, Regenhardt, & Reinheimer, 2002), which requires the selection of resistant strains.
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Table 2 Growth of L. plantarum strains in liquid media (MRS broth, 37 C) in the presence of preservatives used in the dairy industry after 24 h incubation. Preservative
Conc. (%, w/v)
Sodium chloride
3 6 8 10
L. plantarum strainsa RL19
RL22
RL23
RL34
RL36
RL37
RL38
88 63 38 3
89 58 27 9
98 65 32 8
RL29 87 56 30 9
91 63 35 5
89 62 29 9
91 58 25 5
93 57 21 14
Potassium sorbate
0.1 0.2 0.5 1
114 111 85 46
95 83 49 40
93 78 51 33
99 72 56 41
93 69 47 27
94 75 42 26
91 68 43 31
92 65 40 27
Calcium propionate
0.3 0.6
106 120
103 101
106 104
99 97
102 98
102 96
103 100
106 101
Nisaplin
0.01 0.02
52 6
80 9
89 12
93 12
95 45
92 30
87 16
95 43
a
Growth was expressed as the percentage of optical density of control culture (without preservative).
All tested L. plantarum strains were able to grow in the presence of nisin in technologically applied concentrations (Table 2). Only the strains RL34 and RL38 were resistant to higher concentration (0.2 g kg1) and could be used in preserved dairy products. Since L. plantarum RL34 also was shown to have a broad spectrum of antibacterial activity (Georgieva et al., 2008) the strain was selected to study viability in a commercial cheese process. 3.5. Viability of L. plantarum RL34 in cream cheese during the storage and effect of potassium sorbate The viability of L. plantarum RL34 was monitored during three months storage at 4 C in industrially prepared samples of cream cheese with 0.2% (w/w) potassium sorbate and in cream cheese without any preservative (Fig. 3). The effect of preservative and the storage temperature was statistically evaluated. No significant difference (p > 0.05) in the viable cell counts at the end of manufacturing process and after one week was detected. Both types of cheese contained >9 log cfu mL1. After 14 days the population levels decreased to 8.1 and 7.9 log cfu g1 for the cream cheese without and with potassium sorbate, respectively. This reduction was similar for both types of cheese, probably due to the effect of low temperature only (Fig. 3). A slight inhibitory effect in the presence of preservative was measured after the first month (p < 0.05). Nevertheless, at the end of the storage time (3 months), L. plantarum RL34 retained a high number of 107 cfu g1, in both variants of cheese, which is in accordance with the International Recommendations for probiotic and starter cultures in milk
6.0
9 ** ** ** **
8
**
* **
**
** ** *** ***
5.6
4. Conclusion
5.2
The present study estimated technologically relevant properties of eight candidate probiotic L. plantarum strains, isolated from cheeses. The results obtained at a laboratory scale are a good estimation of the tolerance of the selected strains to industrial conditions. Their capacity to survive over extended shelf-times at refrigerated temperatures, their growth viability in the presence of preservatives widely used in food processing, in combination with variable acidifying and coagulation ability and enzyme activity make them appropriate for diverse food applications and highlight on their capability to be incorporated in dairy products. The previously selected candidate probiotic strain L. plantarum RL34 with its b-galactosidase, aminopeptidase, proteolytic and low
7
pH
10
L.plantarum (log cfu g-1)
products (Food Standards Agency, 2002). Some authors discussed the incorporation of probiotic bacteria in cheese as a reliable alternative to the problem of the survival until consumption (Stanton et al., 1998, Vinderola, Prosello, Ghiberto, & Reinheimer, 2000). There are contradictory reports about the effect of sorbate on LAB. Depending on the concentrations, the media used and microorganisms tested it could inhibit or stimulate the growth of LAB strains (Turantas¸ et al., 1999). In our study, potassium sorbate at a concentration of 0.2% did not influence the viability of L. plantarum RL34 (Fig. 3). Thus, this preservative will not prevent the implementation of RL34 in cream cheese, as adjunct probiotic culture. The microbial analysis on Sabouraud agar showed absence of yeast growth in both variants of cheeses at the end of the storage period. In the control-preserved commercial cheese, produced without L. plantarum adjunct, a contamination with yeast (30 cfu g1) was detected after 2 months. The pH of both cheese variants gradually decreased from 5.5 to 4.9 up to 14 days and remained constant till the end of the storage period (Fig. 3). Cheese samples, without addition of L. plantarum, retained the initial pH of 5.5. Lactobacillus strains with low acidifying activity are good as adjunct cultures in soft and semi hard cheeses (Briggiler-Marco et al., 2007). Continued acidifying activity during ripening, reported for strains mainly in soft cheeses, affects the quality of the products. The incorporation of L. plantarum RL34 produced more intensely flavoured cheese (at 3 months) than the commercial control, and the sensory quality of the cheese was generally considered as good. However, the impact of this strain on the sensory profile and flavour development in cream cheese will be the object of further studies.
4.8 6 4.4
5
4.0
4 0
1
7
14
21
30
40
60
90
Time (day) Fig. 3. Viability of L. plantarum RL34 (histograms) and pH change (symbols) in nonpreserved ( , -) and preserved (with 0.2% potassium sorbate; , ) cream cheese during cold storage. Values are means (SD) of three replicate evaluations; t–test: *p < 0.05; **p < 0.01; ***p < 0.001.
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