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In this work, the suitability of Ricotta cheese as a food carrier for functional ingredients was evaluated. The probiotic strain Lactobacillus paracasei subsp. paracasei F19, inoculated at a concentration of 109 cfu/serving size, maintained high counts during the cold storage of Ricotta cheese (7 days at 5°C), without altering the nutritional and sensorial properties of Ricotta samples. Similarly, the addition of 3 % inulin did not significantly change the sensory profile of the cheese, whereas the addition of chestnut flour lowered the perceived sensory characteristics. The synbiotic formulation (with 3 % inulin and 109 cfu/serving size of Lb. paracasei subsp. paracasei F19) altered the Ricotta sensorial characteristics, mainly for an excessive acidification.
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Agro FOOD Industry Hi Tech - vol 24(6) - November/December 2013
Production of functional
Ricotta Cheese
SERENA NIRO, MARIANTONIETTA SUCCI*, LUCIANO CINQUANTA, ALESSANDRA FRATIANNI,
PATRIZIO TREMONTE, ELENA SORRENTINO, GIANFRANCO PANFILI
*Corresponding author
DiAAA, Università degli Studi del Molise, Via de Sanctis snc, 86100 Campobasso, Italy
INTRODUCTION
Functional foods contain one or more components, such as
probiotics and prebiotics, which present the potential to
promote the health of the consumer through mechanisms
not foreseen in conventional nutrition. Probiotics are defined
as viable microorganisms that, when consumed in adequate
amounts (106 to 109 viable cells per day), are beneficial to
the host (1-3). Among their features, probiotic
microorganisms must be normal inhabitants of the human
intestinal tract, must survive passage through the upper
digestive tract in large numbers, and have beneficial effects
when in the intestine. Lactic acid bacteria (LAB) fulfil a
number of the outlined criteria for the selection of probiotic
strains, including the human intestinal origin, the ability to
tolerate acid and bile salts, the low presence of mobilisable
antibiotic resistances, as well as the adaptability to
technological processes for cheese manufacture (4, 5). It
was also shown that strains of Lb. paracasei subsp. paracasei
are able to grow in the presence of different prebiotics (6-8),
defined as non-digestible food ingredients that beneficially
affect the host by selectively stimulating the growth and/or
the activity of one or a limited number of bacteria in the
colon (9). However, a previous investigation highlighted that
the ability of probiotic LAB to use different prebiotics is a
strain-specific character (10). Different types of
carbohydrates can be classified as prebiotics, including
inulin-type fructans, trans-galactooligosaccharides, and
lactulose (9). One of the most frequently used prebiotic is
inulin, a non-digestible fructan which offers an interesting
combination of nutritional properties and technological
benefits (11, 12). This prebiotic is a natural non-digestible
storage polysaccharide consisting of a chain of fructose
molecules with a terminal glucose molecule. It is found in
many vegetable products, amongst which chicory roots are
considered most suitable for industrial applications. Inulin has
the capacity to be a fat substitute, bulking agent, low-
calorie sweetener, and texture modifier (13).
Recent studies showed the possibility to use chestnut based
products as a good substrate for the growth of LAB (14). The
interest in chestnut fruits is due to several functional features,
including their gluten free nature, their low fat content, and
the presence of some phenolic acids (e.g. gallic and ellagic
acid) having antioxidant effects and anti-inflammatory
properties (15). Moreover, chestnuts are mainly composed
by starch (amylose 33 % and amylopectin 67 %), which
provides positive health effects on gut functions thanks to
the bacterial catabolism on amylopectin-derived dextrins
into short-chain fatty acids (15).
The dairy sector is the largest functional food market
accounting for nearly 33 % of the broad market (16).
Different functional dairy products are currently proposed,
such as cheeses (17, 18), yogurts, and other fermented and
probiotic milks (19-24), yog-ice creams and ice cream (25,
26), starch-based dairy desserts (27), sour cream, and butter
cream (28). The success of dairy functional foods can be
explained by the general positive image received by
consumers, linked to the perception of wellness (functional
components able to improve wellbeing), health (chemical
and additive free nature of products), and freshness
(products kept at refrigeration temperatures and
characterized by a relatively short shelf life). Considering
the increase in demand for new functional foods, in this
study we evaluated the possibility to use Ricotta cheese, a
traditional Italian whey protein cheese, as delivery vehicle
for probiotics and prebiotics. Ricotta cheese is an
unripened, creamy dairy product obtained by heat
induced coagulation of whey protein, made mainly from
sheep or goat milk, but also from cow and buffalo milk (29).
In particular, Ricotta can be made only from whey, or from
mixtures of whey and milk (30).
In the light of previous considerations, the aim of this study
was to assess the effect of the addition of a probiotic Lb.
paracasei subsp. paracasei strain and of two different
prebiotics, i.e. inulin and chestnut flour, into Ricotta cheese.
The survival of the probiotic strain as well as the sensory,
chemical and physical characteristics of the Ricotta cheese
were evaluated during the storage at refrigeration
temperature.
MATERIALS AND METHODS
Functional ingredients for Ricotta cheese-making
The probiotic strain Lactobacillus paracasei subsp. paracasei
F19 was isolated by a pharmaceutical formulation (SIFFRA,
Florence, Italy). The strain, revitalised in MRS agar (Oxoid,
Unipath, Basingstoke, UK) at 37°C, was then preserved in the
same medium at 4°C until its use. The inulin (Fibruline) was
obtained from Cosucra s.a. (Fontenoy, Belgium).
Dairy Ingredients
KEYWORDS: Ricotta, whey cheese, prebiotic, probiotic, synbiotic
ABSTRACT: In this work, the suitability of Ricotta cheese as a food carrier for functional ingredients was evaluated. The
probiotic strain Lactobacillus paracasei subsp. paracasei F19, inoculated at a concentration of 109 cfu/serving size,
maintained high counts during the cold storage of Ricotta cheese (7 days at 5°C), without altering the nutritional and
sensorial properties of Ricotta samples. Similarly, the addition of 3 % inulin did not significantly change the sensory profile of
the cheese, whereas the addition of chestnut flour lowered the perceived sensory characteristics. The synbiotic formulation
(with 3 % inulin and 109 cfu/serving size of Lb. paracasei subsp. paracasei F19) altered the Ricotta sensorial characteristics,
mainly for an excessive acidification.
Mariantonietta
Succi
56
Agro FOOD Industry Hi Tech - vol 24(6) - November/December 2013
57
Dairy Ingredients
of each Ricotta sample was aseptically transferred into a
sterile stomacher bag, diluted with 90 mL of sterile
physiological solution (NaCl 9 g·L–1) and homogenised for 1
min in a Lab-blender 400 Stomacher (Seward Laboratory,
London, UK). One mL of the first dilution was used to obtain
tenfold serial dilutions, utilised for microbial counts, detailed
as follows:
-Lactic acid bacteria (LAB) were counted on MRS agar
(Oxoid) after 48 h of incubation at 37°C;
-Enterobacteriaceae were enumerated on VRBGA
(Oxoid) after 36 h of incubation at 37°C;
-Total and faecal coliforms were differentiated on VRBA
(Oxoid) after 36 h of incubation at 37°C and 44°C,
respectively;
-Yeasts and moulds were detected on YPD (36) after
72 h of incubation at 25°C;
-Total bacteria were counted on PCA (Oxoid) after
48 h at 28°C.
Sensory assessment was conducted by a semi-trained panel
composed by 10 judges. Five terms were used: colour, taste
pleasantness, consistence, odour pleasantness, and
homogeneity, with a scale from 1 (poor) to 5 (excellent).
Shelf-life test
Each batch of Ricotta cheese consisted of 9 samples.
After 0, 2 and 7 days of preservation at 5±1°C, the
analyses described above were performed. Moisture, fat,
protein, carbohydrates, ash and fibre were determined
only at the beginning of the storage, whereas sensory
assessment was conducted at 0 and 7 days. Analyses
were performed in triplicate.
Statistical analysis
The analysis of variance (ANOVA) was applied to the data.
The least significant differences were obtained using an LSD
test (P<0.05). Statistical analysis was performed using an SPSS
version 13.0 for Windows (SPSS Inc., Chicago, IL, USA).
RESULTS AND DISCUSSION
Ricotta samples were characterised to assess the most
common quality indexes. For all samples, no significant
variation of composition during storage was evidenced
(data not shown). As shown in Table 1, the composition of
Ricotta cheese added with Lb. paracasei subsp. paracasei
F19 (RLB) was similar to that of the control (RC).
In samples enriched with inulin and chestnut flour (RI, RCF,
RI+LB and RCF+LB), the presence of fibre was evidenced,
whereas all the other components showed lower values in
comparison with those of the control (RC). Only the
glucidic fraction was higher in samples enriched with
chestnut flour (RCF and RCF+LB) (Table 1). In order to
observe fermentative activities on natural (lactose) or
added (starch and inulin) carbohydrates, the content of
The polymer, extracted from chicory roots, had a degree of
polymerization from 2 to 50 and an average degree of
polymerization of 9. The chestnut flour was obtained by
milling white chestnut (Perrotta, Montella, Italy).
Production of functional Ricotta cheese
Three Ricotta cheese-making trials for each experimental
group were conducted on laboratory scale. All the
operations guaranteed the sterility and hygiene of the
product. The whey, collected after the production of pasta
lata cheese from cow milk (Barone farm Vinchiaturo CB,
Italy), was mixed with 10 % milk (v/v) previously heated
to 45°C. Salt was then added to 0.1 % (w/v), and heating
was carried out up to 80-85°C. At this stage a solution of
citric acid (0.11 g·L–1) was added and a gentle stirring was
provided. Following coagulation, the Ricotta cheese was
left to stay for 20 min, picked up and homogenised for 1 min
(Bimby Vorwerk). During homogenisation, prebiotics and/
or probiotics were added. In detail, inulin or chestnut our
were added to achieve 3 g·per serving size (about 100 g).
The probiotic culture of Lb. paracasei subsp. paracasei
F19 was added to reach approximately 109 cfu 100 g–1.
To prepare the microbial culture, cells were overnight
revitalised in MRS broth (Oxoid) at 37°C, harvested by
centrifugation (5000 rpm for 10 min), washed twice in a
solution of 0.9 g·L–1 NaCl and re-suspended in 10 mL of
sterile skimmed milk (Oxoid) at 37°C.
Ricotta samples were then aseptically packaged in sterilised
glass jars with a cap and then cooled to 5±1°C for 7 days
(Figure 1).
The description of Ricotta samples is summarised into this
plan:
- RC: Ricotta cheese without the addition of functional
ingredients and used as control;
- RI: Ricotta cheese added with 3 g 100 g–1 of inulin;
- RLB: Ricotta cheese added with 109 cfu 100 g–1 of Lb.
paracasei subsp. paracasei F19;
- RI+LB: Ricotta cheese added with 3 g 100 g–1 of inulin
and 109 cfu 100 g–1 of Lb. paracasei subsp. paracasei
F19;
- RCF: Ricotta cheese added with 3 g 100 g–1 of chestnut
flour;
- RCF+LB: Ricotta cheese added with 3 g 100 g–1 of
chestnut flour and 109 cfu 100 g–1 of Lb. paracasei
subsp. paracasei F19.
Ricotta cheese analyses
Moisture, fat, protein, and ash were determined following
Official procedures (31). Dietary fiber was quantified by the
enzymatic gravimetric procedure (32). Carbohydrates were
determined by difference. Lactose (33), L-lactic acid (34)
and fructans (35) were determined by using enzymatic kits
(Megazyme International, Ireland), following the
manufacturer’s instructions. For microbiological analyses,10g
Figure 1. Flow sheet of Ricotta cheese processing.
Different letters within the same row indicate significant differences
(P<0.05).
RC: Ricotta cheese, RI: Ricotta cheese + inulin, RCF: Ricotta cheese
+ chestnut flour; RLB: Ricotta cheese + Lb paracasei ; RI+LB: Ricotta
cheese + inulin and Lb paracasei; RCF+LB: Ricotta cheese +
chestnut flour and Lb paracasei.
Table 1. Composition of Ricotta cheeses at 0 days (values reported
in g 100g-1).
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Agro FOOD Industry Hi Tech - vol 24(6) - November/December 2013
Dairy Ingredients
L-lactic acid, lactose and fructans during the storage was
determined in all samples (Table 2).
After 2 days of storage no significant differences were
observed for fermentative activities (data not shown). At 7
days of storage the content in L-lactic acid increased only
in Ricotta samples added with Lb. paracasei subsp.
paracasei F19 (RLB, RI+LB and RCF+LB). The highest
increase in L-lactic acid was noted in RI+LB, underlining the
suitability of free inulin as a substrate for the growth of the
probiotic bacterium used in this research. In fact, in the
samples from the batch RI+LB the content of fructans
(inulin) decreased from 1.93 g 100 g–1 to 1.56 g 100 g–1,
confirming the use of free inulin by the added
microorganism. Moreover, this datum confirms previous
results, which evidenced not only the ability of Lb.
paracasei subsp. paracasei F19 to grow in presence of
inulin, but also the preference of this prebiotic in respect to
other more easily assimilable carbohydrates, such as
lactose (10). On the other hand, during the storage period
lactose decreased significantly (c.a. 10 %) only in the
synbiotic sample RCF+LB, whereas the percentage of
fructans resulted unchanged. Interestingly, the energy
produced by the fermentative activity on carbohydrates,
with the consequent production of lactic acid, was used
by Lb. paracasei subsp. paracasei F19 for the response to
stress conditions, such as low temperature and high solute
concentration (37). In fact, in the experimental probiotic
(RLB) and synbiotic (RI+LB and RCF+LB) samples, the load
of Lb. paracasei subsp. paracasei F19, inoculated at 109
cfu/serving size (100 g), remained constant during the
entire storage period (Figure 2).
LAB resulted undetectable in Ricotta cheese used as
control (Figure 2), and undesirable microorganisms
(Enterobacteriaceae, total and faecal coliforms, and
Eumycetes) were undetectable in all the samples (data
not shown).
The high survival of the probiotic strain during the cold
storage represents an interesting result, since the beneficial
effects of probiotics is strictly related to the daily size of
viable cells ingested (38). Taking into account that dairy
probiotic products must provide not only the minimum
number of cells to confer health effects, but also sensory
acceptance by consumers, sensory attributes of the
functional Ricotta cheese at 0 and 7 days of storage were
investigated (Figure 3).
At the beginning of the storage (Figure 3a), Ricotta cheese
produced with Lb. paracasei subsp. paracasei (RLB) and
with inulin (RI and RI+LB) was particularly appreciated by
judges for all the assayed descriptors, as highlighted by
scores similar to those of the control (RC). On the contrary,
the addition of chestnut flour lowered the perceived
sensorial profile of Ricotta cheese for colour, taste
pleasantness, consistence and homogeneity. After 7 days
of storage (Figure 3b), the sensory profile of Ricotta cheese
resulted substantially unchanged, except for a
considerable decrease (P<0.05) for taste pleasantness in
batch RI+LB due to an excessive acidification, as previously
highlighted by chemical analyses (Table 2).
CONCLUSION
Nutritional benefits associated with the intake of probiotic
microorganisms, soluble dietary fibres or both, could bring
attractive new products to fulfil market niches. The probiotic
strain Lb. paracasei subsp. paracasei F19 showed a high
survival during the cold storage of Ricotta cheese,
maintaining counts over 109 cfu/serving size, without altering
the nutritional and sensorial properties of Ricotta samples.
Therefore, with a standard Ricotta dietary intake (about 100
g), it is possible to obtain a temporary intestinal colonization
by this probiotic. Also, the consumption of 100 g of Ricotta
cheese added with 3 % inulin allows the coverage of 50 %
Figure 2. Counts of lactic acid bacteria in control (RC), probiotic
(RLB) and synbiotic (RI+LB; RCF+LB) Ricotta samples stored at 5°C
for 7 days.
RC: Ricotta cheese, RI: Ricotta cheese + inulin, RCF: Ricotta cheese
+ chestnut flour; RLB: Ricotta cheese + Lb paracasei ; RI+LB: Ricotta
cheese + inulin and Lb paracasei; RCF+LB: Ricotta cheese +
chestnut flour and Lb paracasei.
Figure 3. Sensory profile of the Ricotta samples at 0 (a) and 7 (b)
days of storage (colour and taste have a scale from1 to 5,
consistence from 1 to 4, flavour, and homogeneity from 1 to 3.)
Different letters and numbers within the same column indicate
significant differences (P<0.05).
RC: Ricotta cheese, RI: Ricotta cheese + inulin, RCF: Ricotta cheese
+ chestnut flour; RLB: Ricotta cheese + Lb paracasei ; RI+LB: Ricotta
cheese + inulin and Lb paracasei; RCF+LB: Ricotta cheese +
chestnut flour and Lb paracasei.
Table 2. Evolution of L-lactic acid, lactose and fructans in Ricotta
samples during storage (values reported in g 100g-1).
fibre intake (about 5 g/die), the daily amount indicated to
obtain beneficial effects on the gut. On the other hand, the
synbiotic formulation with inulin and Lb. paracasei subsp.
paracasei F19, as well as that produced with the addition of
chestnut flour, significantly altered the Ricotta sensorial
characteristics. However, it should be stressed that all the
functional products proposed herein can be a basis for the
creation of desserts with Ricotta, whose sensorial
characteristics can be improved and possibly enhanced by
the addition of other ingredients, such as cocoa, coffee,
and fruit syrups.
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... Questa discrasia tra le cariche microbiche effettive e quelle dichiarate è stata osservata anche in un precedente studio (Succi et al., 2014). Tuttavia, in questi ultimi due preparati le cellule vitali sono risultate in numero comunque elevato, ovvero superiore a 10 9 UFC/dose, quantità sufficiente a garantire una temporanea colonizzazione del colon (Di Criscio et al., 2010;Niro et al., 2013). L'analisi DGGE ha confermato per tutte le preparazioni analizzate la presenza unicamente della specie dichiarata in etichetta (dato non riportato). ...
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