Probiotic Soft Sheep's Cheese: Evaluation of Probiotic Survival and Its Influence On Proteolysis and Organoleptic Characteristics
Probiotic Soft Sheep's Cheese: Evaluation of Probiotic Survival and Its Influence On Proteolysis and Organoleptic Characteristics
Probiotic Soft Sheep's Cheese: Evaluation of Probiotic Survival and Its Influence On Proteolysis and Organoleptic Characteristics
*Corresponding author.
Email: fcuffia@unl.edu.ar
400 Cuffia et al./IFRJ 25(1): 399-407
Grattepanche et al. (2008) reported that in Cheddar Institute (Industrial Lactology Institute), where it
type cheese added with bifidobacteria, it significantly was kept frozen at -20°C until its use. On each
increased the production of acetic acid. cheese making day, 40 L of raw milk were unfrozen
On the other hand, even though there are a and pasteurized at 65°C for 20 minutes, cooled to
few studies about the effects on sheep cheeses 39°C (temperature of coagulation) and divided into
manufactured with probiotic strains (Corbo et al., three vats. Starter and probiotic cultures, previously
2001; Albenzio et al., 2010); it has been reported resuspended in 100 mL of sterile milk, were added
that the inclusion of appropriate amounts of at a concentration of 107 CFU mL-1 of milk. Three
bifidobacteria and lactobacilli produces increased batches of soft sheep cheeses were made comprising
levels in proteolysis and lipolysis, which could a control (QT) and two different probiotic cheeses
improve the sensory characteristics (Santillo et al., added with Bifidobacterium animalis subsp. lactis
2009). Additionally, in Argentina there have been BB-12 (QBb) and Lactobacillus acidophilus
reported interesting and promising results about the LA-5 (QLa), respectively. After 10 min, chymosin
incorporation of commercial probiotic strains in sheep produced by fermentation of Aspergillus niger var.
cheese (Cuffia et al., 2012) and in the optimization of wamori (Chy-Max, Inc. Chr Hansen, Denmark. 183
technology for its production thereof (Cuffia et al., IMCU/mL) was added in a suitable dose to obtain
2015 ). In this sense, the selection of probiotic strains the proper firmness for cutting the curd. After 15-20
and determination of their concentration to add are min., the curd was cut to the appropriate grain size
vital for the development of new probiotic cheeses (approximately 25 mm). After 15 min, the mixture
with differential sensory characteristics. Accordingly, was stirred gently to achieve proper moisture. Then,
the aim of this study was to evaluate the survival of two the whey was removed and the curd was placed into
probiotics strains, Lactobacillus acidophilus (LA-5) molds, and kept in a warm chamber (40°C - 3h) until
and Bifidobacterium animalis subsp. lactis (BB-12) reaching pH 5.10 ± 0.05. Afterwards, the cheeses
and its influence on the composition, proteolysis and were held in a conditioning chamber at 4°C and 92%
sensory characteristics of a fresh sheep cheese. relative humidity (with air circulation at reduced
The aim of this work was to evaluate the survival speed to avoid excessive surface evaporation), for 24
of two commercial strains of probiotic bacteria: h. This stage has the purpose of regulating the cooling
Bifidobacterium animalis subsp. lactis BB-12 and rate and allows the development of fermentation to
Lactobacillus acidophilus LA-5, and their influence compensate for the increase in pH derived from the
on the composition, proteolysis and sensory salts balance, which occurs during brining (Cuffia et
characteristics of soft sheep cheese. al., 2011). Approximately 4 kg of cheese obtained,
was divided into eight portions of 500 g and salted
Materials and Methods by immersion in brine at 15% p/w, as established in
a previous work (Cuffia et al., 2015). After salting,
Starter and probiotic cultures the cheeses were placed in the same conditioning
A cheese starter culture consisting of chamber (4°C and 92% relative humidity), and on
Streptococcus salivarius subsp. thermophilus ST- the fourth day they were packed under vacuum in
M5 (Chr. Hansen, Inc., Denmark) and the probiotic shrink plastic bags, until completing maturation.
cultures of Bifidobacterium animalis subsp. lactis Three replicates of cheeses were made on successive
BB-12 and Lactobacillus acidophilus LA-5 (Chr. cheese-making days.
Hansen, Inc., Denmark) were used as cultures
(freeze-dried) in cheesemaking experiments. Both Survival of bacteria in cheeses
commercial probiotic cultures from Chr. Hansen have Viability of starter and probiotic bacteria was
probiotic functions which have been demonstrated in assessed during production (in inoculated milk
several works: Shioya et al. (2000), Sheu et al. (2002), and acidified curd), and in cheeses during ripening
Chouraqui et al. (2004) and Pitkälä et al. (2007) for at 4, 15 and 30 days according to Vinderola et al.
B. animalis BB-12; and Shioya et al. (2000) and (2003). Starter bacteria was enumerated on M17
Sheu et al. (2002), for Lb. acidophilus LA-5. agar (Biokar Diagnostics, Beauvais, France) after
incubation under microaerobic condition at 37°C
Cheesemaking for 48 h. The counts of Bifidobacterium animalis
Raw sheep milk, provided by the School of subsp. lactis were determined on MRS agar with
Agriculture, Farm and Livestock from Litoral addition of 0.1% (w/v) cysteine (Biopac - Buenos
National University (EAGyG-UNL), was refrigerated Aires, Argentina) (MRSc), after incubation undrer
and transported at 4ºC to the pilot plant of our anaerobic conditions (AnaeroPack®- Anaero,
Cuffia et al./IFRJ 25(1): 399-407 401
Mitsubishi Gas Chemical CO., INC., Tokyo, Japan) Laboratories, California, USA) by Andrews (1983)
at 37°C for 48 h. Lactobacillus acidophilus was method, with a concentration of acrylamide of 7.5%.
enumerated on MRS agar, with addition of 0.1% Proteins were stained by Coomasie blue G-250.
(w/w) sorbitol, after incubation under anaerobic
conditions (AnaeroPack®-Anaero, Mitsubishi Gas Sensory analysis
Chemical CO., INC., Tokyo, Japan) at 43°C for 72 h. Descriptive sensory analysis was performed at
the end of the ripening (30 days). The three cheeses,
Gross composition and pH identified by random numbers, were presented
Cheese samples were grated and analyzed in simultaneously to each evaluator. The panel was
duplicate for fat matter by Gerber van Gulik method composed of eight participants trained in the subject,
(FIL - IDF Nº 152 A 1997), moisture by oven-drying who, using unstructured scales anchored at the ends,
at 102°C (AOAC 926.08, 1990), and total protein evaluated in two separate sessions the following
by Kjeldahl method (AOAC 920.123, 1990) using attributes: odor, color, appearance of mass, elasticity,
a Digestion System 6 (1007 Digester, Tekator, mouthfeel, cream flavor, salty taste, bitter taste, acid
Switzerland) and BÜCHI Distillation Unit B-324 taste and residual flavor.
(Sweden). The pH of cheese was measured according
to Bradley et al. (1993), with a pH meter (Orion Statistical analysis
Research Incorporated, United States) introduced The results were processed by analysis of
in slurry prepared by blending a mix 1:1 of grated variance (ANOVA) using Statgraphics Plus v3.0
cheese in H2O. Cheese composition and pH were software (Statistical Graphics Corp.) to determine
analyzed at day 4, 15 and at the end of ripening (30 the effect of the addition of probiotic bacteria on
days). gross composition, pH, proteolysis and sensory
characteristics of cheeses. When significant
Soluble nitrogen (SN) differences were presented (p<0.05), Duncan’s test
Cheese samples were treated to obtain crude was applied to detect homogeneous groups of means,
citrate extract and soluble fractions at pH 4.6 (SN using the same software.
4.6), in TCA 12% (SN TCA) and PTA 2.5% (SN
PTA), according to Hynes et al. (2003). The crude Results
cheese extract was obtained by adding 20 mL of 0,5
M sodium citrate to 10 g of cheese and grounding Cheese composition during ripening
to homogeneity using a pestle. Deionized water was Values of pH were similar in control and probiotic
added to ~90 mL, and the pH was adjusted to 4.6. cheeses at each sampling day (Table 1). Nevertheless,
After centrifugation (3000 x g / 15 min), the soluble in all cheeses, pH increased significantly along the
fraction volume was adjusted to 100 mL. The TCA ripening. On the other hand the levels of moisture,
12% and PTA 2.5% soluble fractions were obtained fat matter and total protein were also similar between
from 4.6 soluble fraction according to Gripon et al. control and probiotic cheeses, which demonstrate that
(1975). The N content was determined in duplicate the cheese making was reproducible. Furthermore,
by the macro-Kjeldahl method according to the IDF the values of moisture were established according
method (IDF, 1993). to the Argentinean legislation (Código Alimentario
Argentino).
Electrophoresis
The insoluble residue at pH 4.6 was purified. In Survival of bacteria during manufacture and repining
order to do that, samples were re-dissolved by adding of soft sheep cheeses
200 mL of distilled water and bringing the pH to 7 with Microbiological counts of starter bacteria
stirring. After being kept for about 10 min under these increased approximately one log order in the curd
conditions, the insoluble residue was re-precipitated regarding its concentration in milk and were kept in
at pH 4.6, proceeding in the same manner as in the a range of 8 log10 throughout ripening time (Table 2).
extraction. This operation was repeated twice. Finally, In addition, starter population was similar in control
the insoluble residue was washed with distilled water and experimental cheeses, demonstrating that the
twice (by suspension and centrifugation). Samples addition of probiotic bacteria in experimental cheeses
thus obtained were preserved in a freezer at -18°C did not affect starter viability.
for subsequent electrophoretic analysis.
Electrophoretic assessment was carried out by Proteolysis assessment
Urea-PAGE in a Mini-Protean II cube (BioRad The nitrogen content in the different soluble
402 Cuffia et al./IFRJ 25(1): 399-407
Table 1. pH and gross composition of control (QT) and probiotic (QBb and
QLa) soft sheep cheeses during ripening
Table 2. Viable counts (Log10 CFU/g) of primary starter and probiotic bacteria during
cheesemaking (milk and curd) and ripening of control and probiotic soft sheep cheeses.
all ripening time (Vinderola et al., 2003; Bergamini those of control cheese. Likewise, Ong et al. (2006)
et al., 2009; Burns et al., 2012). In general, cheeses showed an increase of the fraction of SN PTA after 4
show great advantages over other fermented dairy months of ripening in Cheddar cheese with probiotic
products like yogurt as a vector for incorporating strains of Lb. acidophilus, Lb. casei, Lb. paracasei,
probiotic bacteria to diet because they have a and bifidobacteria.
higher pH and buffer capacity, higher consistency In previous works, we found that different strains
and more fat content (Ong and Shah, 2009). These of probiotic bacteria produced a different impact on
characteristics would be able to offer more protection secondary proteolysis of probiotic semihard cheeses,
to probiotic bacteria during storage and transit in the made with cow milk. In particular, Lb. acidophilus
gastrointestinal tract (Gardiner et al., 1998). produced the higher effect with an increase in the
The nitrogen content in the soluble fraction at levels of free amino acids and the fraction of SN PTA,
pH 4.6 represents the primary proteolysis, and it while Lb. paracasei only influenced on the levels of
is produced from the breakdown of intact caseins a few amino acids and Bb. lactis did not produce
(especially αs1 and β caseins) mostly by non- any change in the proteolytic process (Bergamini et
microbial proteases. In soft cheeses, in particular, al., 2009). The differences found in the proteolytic
residual rennet is the main non-microbial agent activity of probiotic bacteria can be attributed to
which participates in proteolysis during ripening the heterogeneity in their peptidolytic potential, and
(Delacroix-Buchet and Fournier, 1992; Vélez et al., actual activity of the enzymes in the cheese matrix,
2015). which are largely strain-dependent (Corbo et al.,
On the other hand, the nitrogen content in the 2001; Ong et al., 2007; Gómez et al., 2010; Albenzio
SN TCA and SN PTA represents the secondary et al., 2013b).
proteolysis, which is mainly produced by the activity The Urea-PAGE results suggested that, in
of microbial proteolytic enzymes from starter general, αs1 casein underwent a limited hydrolysis,
and adjunct cultures and also from NSLAB (non- mainly by the residual rennet, releasing the typical
starter lactic acid bacteria) (Fox et al., 1996). These peptide αs1-I casein, which was more noticeable at
enzymes hydrolyze large and medium peptides 30 days of ripening (Irigoyen et al., 2002).
leading to the production of smaller peptides and free Sensory analysis is very useful to know the
amino acids. The compounds contained in SN TCA organoleptic properties of the cheese through the
are medium-sized to small peptides, amino acids and senses. It is fundamental to determine the acceptability
smaller nitrogen compounds, such as amines, urea of the product by the consumer, this constituting a
and ammonium, whereas SN PTA fraction contains very important parameter for marketing the product
very small peptides, amino acids and smaller N (Carpenter et al., 2000). Appearance and mouthfeel
compounds except dibasic amino acids and ammonia attributes are important drivers of consumer liking,
(Ardö, 1999). The addition of both probiotic strains as they can affect choice behavior (Wadhwani
produced a significant impact on the secondary and McMahon, 2012). These results demonstrate
proteolysis but only on the levels of the SN PTA, that both commercial probiotic cultures did not
being this effect higher for Lb. acidophilus. Thus, our produce defects of flavor, and could even improve
results show that these strains, despite not affecting organoleptic characteristics of soft sheep cheeses,
the primary proteolysis, have peptidolytic enzymes apart from providing probiotic characteristics based
which led to an increase in the production of small on the properties of these strains.
peptides and amino acids. Similar to our results, The addition of probiotic cultures should not
Gardiner et al. (1998) and McBrearty et al. (2001) result in lower acceptance of the food compared with
did not detect any influence of probiotic cultures of a similar conventional product (Cruz et al., 2010).
lactobacilli and bifidobacteria on primary proteolysis Some authors have reported the effect of the addition
of Cheddar cheese, evaluated by means of the peptide of probiotic cultures on the sensory characteristics of
profiles obtained by size exclusion HPLC of water- different types of cheeses. In most of these works it
soluble peptides. In addition, other authors also is highlighted that the influence is dependent on the
found some differences in the secondary proteolysis strain (Albenzio et al., 2013a). While some strains
between cheeses with and without probiotic bacteria. produce an improvement in the sensory properties
Corbo et al. (2001) verified that peptide profiles (Minervini et al., 2012), other strains do not
obtained by reversed-phase fast protein liquid produce changes or produce defects in the product
chromatography of the soluble fraction of probiotic (Tungjaroenchai et al., 2001; Sarantinopoulos et
Canestrato Pugliese cheeses (sheep cheeses) al., 2002). Dinakar and Mistry (1994) reported that
containing bifidobacteria were more complex than bifidobacteria added to Cheddar cheese did not affect
Cuffia et al./IFRJ 25(1): 399-407 405
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