freeze-shocked forms of commercial Lactobacillus helveticus. Lh.Bo2 as adjunct culture ... Powdered skim milk (Merck Co) was reconstituted to 11%. (RSM) and ...
doi: 10.1111/j.1471-0307.2011.00720.x
ORIGINAL RESEARCH
Impact of native Lactobacillus paracasei subsp. paracasei and Pediococcus spp. as adjunct cultures on sensory quality of Iranian white brined cheese JAVAD BAROUEI, 1 , 4 * AHMAD KARBASSI, 1 HAMID B GHODDUSI, 2 ALI MORTAZAVI, 3 ROGHAYEH RAMEZANI 1 and MAHTA MOUSSAVI 4 1
Department of Food Science and Technology, School of Agriculture, Shiraz University, Postal Code 71444, Shiraz, Iran, Microbiology Research Unit, Department of Health and Human Sciences, London Metropolitan University, 166-220 Holloway Road, London N7 8DB, UK, 3Department of Food Science and Technology, School of Agriculture, Ferdowsi University, PO Box 91775-1163, Mashhad, Iran, and 4Laboratory of Microbiology, Life Science Building, School of Environmental and Life Science, The University of Newcastle, Callaghan 2308, NSW, Australia
2
Paired wild-type cultures consisting of a Lactobacillus paracasei subsp. paracasei (three strains) or Pediococcus pentosaceus (one strain) and a Pediococcus inopinatus (five strains) were used as adjunct cultures in the production of Iranian white brined cheese. After 8 weeks of ripening, adjunct-treated cheeses produced by L. paracasei subsp. paracasei and P. inopinatus received significantly higher scores for flavour ⁄ taste, aroma, texture and overall preference than those produced by P. pentosaceus and P. inopinatus as well as the control cheese (P < 0.05). In conclusion, a greater improvement of sensory quality of cheeses was strongly associated with the presence of L. paracasei subsp. paracasei rather than pediococci. Keywords Adjunct culture, Lactobacillus paracasei subsp. paracasei, Pediococcus, Sensory quality, Iranian white brined cheese.
INTRODUCTION
the cheese rather than acid (Clark and Agrawal 2007). Primarily, adjunct cultures of NSLAB are added to cheese for contribution to flavour and texture development (El Soda et al. 2000). The common bacterial strains used as adjunct cultures are mainly from non-starter mesophilic lactobacilli especially Lactobacillus paracasei subsp. paracasei and Lactobacillus plantarum and then pedicocci such as Pediococcus pentosaceus and Pediococcus acidilactici and strains of enterococci spp. (Beresford and Williams 2004). L. paracasei subsp. paracasei is likely the best known and most extensively studied adjunct culture used in cheese varieties such as Cheddar, Edam and feta. In a set of studies, amongst adjunct lactobacilli isolated from a high-quality raw milk cheese, L. paracasei subsp. paracasei and L. plantarum were highlighted to contribute to the higher flavour intensity and sensory scores when included in pasteurised milk cheese compared with control cheeses with no adjunct cultures added (McSweeney et al. 1994; Lynch et al. 1996, 1999). It has been reported that L. paracasei subsp. paracasei
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Non-starter lactic acid bacteria (NSLAB) contribute to desired cheese ripening events through their metabolic activities such as proteolysis and lipolysis which influence flavour and texture development of the product (Crow et al. 2001; Beresford and Williams 2004). However, application of the specified and strict hygienic standards in cheese manufacturing process leads to a drastic reduction in the population of favourable NSLAB in cheese (El Soda et al. 2000). To overcome this problem, deliberate addition of selected strains of NSLAB isolated from premium quality raw milk cheese, so-called adjunct cultures either as nonviable (attenuated) or viable (nonattenuated) cells, to the cheese milk has been suggested as a reliable technological approach to improve organoleptic quality of cheese within a reasonable amount of time (El Soda et al. 2000; Fox and McSweeney 2004). Adjunct culture is defined as microbial culture that is added to cheese milk, usually along with starter culture, owing to imparting desirable attributes to
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*Author for correspondence. E-mail: Javad.Barouei@uon. edu.au
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isolated from Dumbo cheese and used as adjunct cultures in a cheese model enhanced sensory quality of the cheese more effectively than other cultures including L. plantarum (Antonsson et al. 2003). Application of a combination of L. paracasei and L. plantarum isolated from raw ewe’s milk as adjunct culture significantly improved flavour in experimental Roncal cheese made from the pasteurised milk earning almost the same sensory scores as those given to cheese made from raw milk (Ortigosa et al. 2005). Pediococcus spp. have also been applied as adjunct cultures in the production of different types of cheeses (Bhowmik et al. 1990; Vafopoulou-Mastrojiannaki et al. 1990; Tzanetakis et al. 1991; Agarwal et al. 2006; Franciosi et al. 2008). Pediococcus pentosaceus has been the most used species of the genus as adjunct culture. Enhanced flavour production in brined cheeses such as feta and Teleme was observed with the incorporation of P. pentosaceus as adjunct culture into the cheese milk (Vafopoulou-Mastrojiannaki et al. 1990; Tzanetakis et al. 1991). The results of another study revealed that using P. pentosaceus NCDO 559 as an adjunct culture significantly enhanced proteolysis during ripening of low-fat Cheddar cheese compared to that of control cheese. Development of a sharp, aged Cheddar flavour was reported in 6-month-old cheese treated with a Pediococcal adjunct culture, whereas control cheese lacked such a flavour (Bhowmik et al. 1990). A multispecies combination of L. paracasei subsp. paracasei, L. plantarum and P. pentosaceus at inoculation level of 103 cfu ⁄ mL was used for reducing variability in Italian Puzzone di Moena cheese production (Franciosi et al. 2008). Iranian white cheese is categorised as a pickled cheese which its ripening process in brine lasts up to 2 months. The end product is characterised by a desired sour and salty taste, white to cream colour and a crumby semi-soft to semi-hard texture. Higher overall sensory quality scores have been reported for a 45-day-old Iranian white cheese treated with unattenuated or freeze-shocked forms of commercial Lactobacillus helveticus Lh.Bo2 as adjunct culture relative to the control cheese. At day 60, adjunct-treated cheeses with the unattenuated culture graded as the best in terms of overall sensory quality followed by the control and cheese treated with the freeze-shocked adjunct culture (Hashemi et al. 2009). To the best of the authors’ knowledge, there have been no previous studies that examined the effects of applying native (wild-type) NSLAB isolated from traditional Iranian raw ewe’s milk cheese as adjunct cultures in the production of Iranian white brined cheese, and knowledge of this strategy may lead to enhance desirable organoleptic quality of the cheese. The objective of this work was to determine the effect of unattenuated adjunct cultures consisting of selected wild L. paracasei subsp. paracasei and pediococci spp strains previously isolated and identified as predominant NSLAB present in a premium quality portion of Salmaas raw ewe’s milk cheese on the sensory characteristics of Iranian white brined cheese made from pasteurised milk. 2011 Society of Dairy Technology
MATERIALS AND METHODS
Bacterial cultures and growth conditions Lactobacillus paracasei subsp. paracasei strains LPP39, LPP46 and LPP52; P. pentosaceus strain PP51 and Pediococcus inopinatus strains PI31, PI41, PI45, PI50 and PI53 all previously isolated from a premium quality Salmaas raw ewe’s milk cheese in our laboratory (unpublished data) were used as adjunct cultures in this study. Bacterial strains had already been stored at )20C in a freezing medium described elsewhere (Mortazavi et al. 2007). Prior to use, the lactobacilli and pediococci were recovered by two consecutive subculturing in MRS medium (Merck Co, Darmstadt, Germany) and overnight incubation under anaerobic conditions (GasPak Systems, BD, Franklin Lakes, NJ, USA) at 37 and 30C, respectively. Determination of fermenting patterns of cultures Carbohydrate utilisation patterns of cultures were analysed using API 50 CH kits (bioMerieux sa, Marcy-I’Etoile, France) as per manufacturer’s instructions. Enzyme profiles of cultures Enzyme profiles of the cultures were determined using API ZYM strips (bioMerieux sa). Overnight grown cultures in MRS broth were centrifuged at 10 000 · g for 15 min at 4C using a Herolab centrifuge model HiCen 18 (Herolab GmbH Laborgera¨te, Wiesloch, Germany). The culture supernatant (CS) was removed and kept refrigerated. Bacterial pellets were then washed twice with 0.1 M phosphate buffer (pH 7.0) and then resuspended in the fresh buffer. Crude cell-free extracts (CCFE) were obtained by sonication of the bacterial suspensions using an ultrasonic processor model XL 2020 (Misonix Inc., Farmingdale, New York, NY, USA) followed by separation of CCFE from other cell fragments by centrifugation as described elsewhere (Habibi-Najafi and Lee 1994). Amounts of 25 lL of CCFE or CS were added to the cupules of API ZYM strips. The strips were then incubated at 37C for 4 h. The assay was then undertaken as per manufacturer’s instructions. Enzyme activity was measured by comparing the colour developed with the chart provided by the manufacturer and expressed on a scale of 0 (no activity) to 5 (maximum activity; ‡40 nM of chromophore released). Measurement of titratable acidity Powdered skim milk (Merck Co) was reconstituted to 11% (RSM) and sterilised (121C for 5 min). Hundred-millilitre aliquots of sterile RSM were dispensed into sterile Erlenmeyer’s flasks, inoculated by 1% of each fresh overnight grown culture, and incubated for 48 h. Acid production was measured after 24 and 48 h by titration with 0.1 N NaOH (Merck Co) to the turning point of phenolphthalein. Titratable acidity was reported as the percentage equivalent of lactic acid. 527
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Assessment of proteolytic activity Proteolytic activity of the cultures was quantitatively assessed in a sterile 11% RSM after 2, 6, 10, 24 and 48 h of incubation using O-phthaldialdehyde and determination of optical density at 340 nm (Church et al. 1983). Preparation of adjunct cultures Bacterial cells were harvested in their stationary phases by refrigerated centrifugation at 2300 g for 10 min and washed thrice with 0.1 M phosphate buffer (pH 7.0). Bacterial pellets were then resuspended in the fresh buffer and vortex-mixed. Each strain of L. paracasei subsp. paracasei strains (LPP39, LPP46 and LPP52) or P. pentosaceus strain PP51 was individually combined with Pediococcus inopinatus strain (PI31, PI41, PI45, PI50 and PI53) to make 20 paired adjunct cultures. Fresh bacterial suspensions were made at the same day of cheesemaking and kept refrigerated until use. Cheesemaking Twenty adjunct-treated experimental Iranian white brined cheeses and a control cheese (without adjunct culture) were manufactured according to the guidelines described by the Iranian standards (Iranian Standards 2005). All cheese portions were manufactured from the same production lot of cows’ milk on a laboratory scale at Fars Pegah Dairy plant (Fars Pegah Dairy Co., Shiraz, Iran). Briefly, cheese milk was standardised to a fat content of 2.5%; HTST pasteurised at 72C for 15 s and cooled down to 34 ± 1C. Then, 100 ppm calcium chloride (Merck Co) and inoculums of 1% mesophilic cheese starter culture G3 Mix 6 (Laboratorium Wiesby GmbH & Co. KG, Niebull, Germany) comprised a blend of strains Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were added to all cheese milk portions. Adjunct cultures were added along with the starter cultures to achieve a population of 1 · 106 cfu ⁄ mL in the cheese milk prior to rennet coagulation using addition of 20 ppm of commercial fungus coagulant Fromase (DSM Food Specialties, Brøndby, Denmark) derived from Rhizomucor miehei. The cheese milk was then mixed for few minutes and left undisturbed at 34–36C for a further 30 min till coagulation was completed. The final coagula were cut and drained using cheese cloth and then pressed for 120 min. Five hundred-gram curd pieces were placed separately in tin cans containing 22% w ⁄ v heat-treated (85C for 5 min) and cooled brine for 8 h. Finally, the cheeses were transferred separately to sterile plastic bags (RWP Flexible Packaging Ltd, Bristol, UK) containing 12% heat-treated brine. The bags were then heat-sealed and stored at 12C in a ripening room for a period of 8 weeks. Chemical analyses All produced cheeses were analysed for compositional and physicochemical parameters. Dry matter content was measured by oven drying at 103–105C according to the AOAC official 528
method 16.192 (AOAC 1980). Total protein content was assessed by Kjeldahl method (Pearson 1973). Logarithm of moisture to protein ratio (Log MPR) for each cheese was then calculated using moisture and protein content data obtained as described above. Fat content was analysed by the Gerber method (James 1996). Percentage of fat content in dry matter (FDM) and moisture in fat-free cheese (MFFC) for each cheese were then calculated using relevant data obtained as described above. Salt content was determined by a potentiometric method (IDF 1988), and the results were expressed as percentage of salt in dry matter (NaCl in DM). pH of cheese was measured by a digital Metrohme pH-meter model 632 (Metrohm AG, Herisau, Switzerland). Titratable acidity (TA) of cheeses was determined according to the AOAC official method 16.022 (AOAC 1980), and results were expressed as percentage of lactic acid in the cheese.
Microbiological analyses The relevant ISO and ⁄ or IDF microbiological standards for microbial analyses of milk and dairy products were used for the enumeration of total coliforms (IDF 1998), Escherichia coli (ISO ⁄ IDF 2005), coagulase-positive Staphylococcus aureus (ISO ⁄ IDF 2003), total salmonellae (ISO ⁄ IDF 2001) and total moulds and yeasts (ISO ⁄ IDF 2004) in cheeses. Sensory evaluation A sensory panel of 17 was screened from 31 volunteers using primary taste and food-related odour recognition tests (Carpenter et al. 2000). Selected panellists were then trained for suitable cheese sensory properties. Panellists were asked to evaluate experimental cheese samples for flavour ⁄ taste, aroma and texture using a nine-point hedonic scale (Moskowitz et al. 2006). The qualitative scores were then converted to numerical scores for computation and statistical analysis purposes; like extremely, 9; dislike extremely, 1 and intermediates from 2 to 8. Overall preference of cheese was calculated using the following formula based on authors’ experience and dairy expert advice: ðTaS 20Þ þ ðArS 10Þ þ ðTxS 15Þ 45 where OP, overall preference; TaS, taste score; ArS, aroma score; TxS, texture score. OP ¼
Statistical analysis A randomised complete block design was used for data analysis. Analysis of variance (ANOVA) was performed using SPSS software version 15 (SPSS Inc., Chicago, IL, USA). Comparison of means was undertaken using Duncan’s multiple range test. A P < 0.05 was considered statistically significant. RESULTS AND DISCUSSION Of 49 carbohydrates examined, 22 carbohydrates were fermented by L. paracasei subsp. paracasei strains, whereas 2011 Society of Dairy Technology
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P. pentosaceus and P. inopinatus strains utilised 15 and 12 carbohydrates, respectively. Twelve carbohydrates including galactose, D-glucose, D-fructose, D-mannose, maltose, lactose, N-acetylglucosamine, esculin, sailicine, cellobiose, b-gentiobiose and D-tagatose were fermented by all individual cultures examined. All L. paracasei subsp. paracasei strains were able to ferment 10 more carbohydrates including glycerol, ribose, mannitol, sorbitol, amygdaline, arbutin, saccharose, trehalose, melezitose and gluconate, whilst P. pentosaceus utilised only three more carbohydrates including ribose, arbutin and trehalose (Table 1). In early stages of cheesemaking, lactose, glucose and galactose, the main carbohydrates of milk, are quickly used up by starter LAB (Williams et al. 2000). NSLAB therefore rely on other potential energy sources available in cheese such as amino acids, fatty acids, organic acids, glycerol or carbohydrates released from components of cheese curd, e.g. glycomacropeptide of j-casein, glycoprotein and glycolipid fractions of the milk-fat globule membrane. The main carbohydrates released are galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid. Moreover, lysed cells of starter bacteria liberate carbohydrates such as ribose, deoxyribose, N-acetylglucosamine, N-acetylmuramic acid which could be utilised by NSLAB (Adamberg et al. 2005). The results of API 50 CH assay revealed that galactose, mannose and N-acetylglucosamine could be utilised by all individual cultures examined. Ribose was used by strains of L. paracasei subsp. paracasei and P. pentosaceus, whilst
glycerol could be fermented only by L. paracasei subsp. paracasei strains. These results are consistent with the previous research showing that some strains of L. paracasei subsp. paracasei are able to ferment ribose, galactose, mannose and N-acetylglucosamine as energy sources (Saxelin et al. 1996; Williams et al. 2000; Adamberg et al. 2005). Utilisation of glycerol by strains of L. paracasei subsp. paracasei has been rarely reported (Collins et al. 1989; Paludan-Muller et al. 1999). Pediococcus pentosaceus strains typically utilise galactose, mannose, ribose and N-acetylglucosamine and variably use glycerol (Caldwell et al. 1996; Holzapfel et al. 2006), whilst P. inopinatus can ferment galactose and mannose but not glycerol and ribose (Holzapfel et al. 2006). In conclusion, L. paracasei subsp. paracasei strains possessed a wider spectrum of glycolytic activity compared with P pentosaceus and P. inopinatus. Enzyme profiles of individual cultures indicated medium to high peptidase (leucine arylamidase, valine arylamidase and cystine arylamidase) activities in the culture supernatant of L. paracasei subsp. paracasei strains and P. pentosaceus. These data are consistent with the previous studies showing strong activities of leucine and valine arylamidase and low activity of cystine arylamidase for L. paracasei subsp. paracasei and P. pentosaceus strains isolated from milk and various milk products including cheese (Ballesteros et al. 2006; Mathara et al. 2004; Sulieman et al. 2006; Tzanetakis and Litopouloutzanetaki 1989). A high leucine arylamidase activity was found in crude cell-free extracts of all P. inopinatus strains.
Table 1 Carbohydrate utilisation profiles of strains used as adjunct cultures in this study
Pediococcus inopinatus
Pediococcus pentosaceus
Lactobacillus paracasei subsp. paracasei
Strain
PI31
PI 41
PI 45
PI 50
PI 53
PP51
LPP39
LPP46
LPP52
Acid from Glycerol Ribose Mannitol Sorbitol Amygdaline Arbutin Maltose Lactose Saccharose Trehalose Melezitose Gluconate
) ) ) ) ) ) + + ) ) ) )
) ) ) ) ) ) + + ) ) ) )
) ) ) ) ) ) + + ) ) ) )
) ) ) ) ) ) W + ) ) ) )
) ) ) ) ) ) + + ) W ) )
) + ) ) ) + + + ) + ) )
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + + + +
Symbols: ), negative; +, positive; W, weak. All Isolates fermented galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, esculin, sailicine, cellobiose, b-gentiobiose and D-tagatose, whereas none of isolates fermented erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, b-methyl xyloside, L-sorbose, rhamnose, dulcitol, inositol, a-methyl mannoside, a-methyl-D-glucoside, melibiose, inulin, D-raffinose, Amidon, glycogen, xylitol, D-turanose, D-xylose, D-fucose, L-fucose, D-Arbitol, L-Arbitol, 2-ceto-gluconate and 5-ceto-gluconate.
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530
1.6 LPP
1.4
PP
OD at 340 nm
1.2
PI
1 0.8 0.6 0.4 0.2 0
0
2
6
10
24
48
Time (h) Figure 1 Proteolytic activity of bacterial strains using o-Phthaldialdehyde spectrophotometric assay during 48 h incubation. For reasons of clarity and because of similar proteolytic activity patterns of L. paracasei subsp. paracasei strains (LPP) and P. inopinatus strains (PI), only one curve is presented for each of these culture groups.
0.7
PI31 PI 45 PI 53 LPP39 LPP52
0.6
Titratable acidity (%)
Only P. inopinatus PI53 exhibited a high valine arylamidase activity and weak cystine arylamidase activity in its crude cellfree extract but not culture supernatant. Medium level of esterase and low level of esterase–lipase activities were found in the culture supernatant of L. paracasei subsp. paracasei and P. pentosaceus. The findings would appear consistent with several studies in which medium level of esterase and low level of esterase–lipase activities were observed in L. paracasei subsp. paracasei strains isolated from cheese and fermented milk products (Mathara et al. 2004; Ballesteros et al. 2006; Sulieman et al. 2006). All strains of P. inopinatus were lipolytic negative, as indicated by esterase C4, esterase–lipase C8 and lipase activities. Strong acid phosphatase activity was noted in culture supernatant of all cultures examined. A low to moderate activity of phosphohydrolase was also found in both culture supernatant and crude cell-free extracts of cultures with the exception of PI45 which was found to be phosphohydrolase negative. With the exception of b-galactosidase, the activity of other glycolytic enzymes was not detected across the bacterial strains examined. A high b-galactosidase activity was found only in crude cell-free extracts of P. inopinatus strains. Specifically, b-glucuronidase and b-glucosidase activities are important in terms of safety of bacterial candidates to be used in food products. These enzymes may implicate in the bioavailability of some toxicants and formation of carcinogens, mutagens and various tumour promoters (Rowland 1991; O’Brien et al. 1999; Saarela et al. 2000). In conclusion, it seemed that all bacterial strains examined did not have the ability to produce harmful enzymes. Furthermore, L. paracasei subsp. paracasei strains and P. pentosaceus showed much more proteolytic and lipolytic activity when compared with P. inopinatus strains. The results of acidifying and proteolytic activities of individual bacterial cultures examined are presented in Figures 1 and 2. Three main bacterial groups were identified with respect to kinetics of milk acidification. L. paracasei subsp. paracasei strains were found to produce the highest amount of acid as much as 0.58% in milk after 48-h incubation. Pediococci strains showed a significantly lower acidifying activity compared with that of L. paracasei subsp. paracasei strains. Amongst Pediococci, a moderate acidifying activity was observed for P. pentosaceus, whilst P. inopinatus strains showed the lowest acidification activity with maximum level of 0.3%. It is apparent from the results of proteolytic activity of the cultures that L. paracasei subsp. paracasei strains were much more active in the degradation of milk proteins than Pediococci spp. All strains of L. paracasei subsp. paracasei indicated a significantly higher proteolytic activity (OD = 1.3) than Pediococci spp (
