INTRODUCTION
Production of functional foods is being recognized as the number one
global food biotechnology industry as changing trends in population demography,
consumer affluence, increased education, life expectancy and improved
healthcare give rise to a rapidly emerging diet and health conscious consumer
clientele (Belem, 1999; Childs, 1999; Dillard and German, 2000). The term
Functional Food was first introduced in Japan in the mid-1980s and refers
to processed food containing ingredients that aid specific bodily functions
in addition to being nutritious (Swinbanks and O`Brien, 1993).
Probiotics are defined as selected, viable microbial dietary supplements
that, when introduced in sufficient quantities, beneficially affect human
organism through their effects in the intestinal tract (Zimmer and Gibson,
1998; Sanders, 1998). They may play an important role in helping
the body protect itself from infection, especially along the colonized
mucosal surfaces of the gastrointestinal tract (Sanders, 2003).
Probiotic bacteria beneficially affect human health by improving the
gut microbiota balance and the defenses against pathogens. Additional
health benefits attributed to probiotics are the stimulation of the immune
system, blood cholesterol reduction, vitamin synthesis, anti-carcinogenesis
and anti-bacterial activities (Heenan et al., 2004). Two other
important criteria to determine the efficacy and the success of the product
containing probiotics are the acceptance of the product by the consumers
and the survival of probiotic microorganisms during its production (Heenan
et al., 2004). In general, the food industry has applied the recommended
level of 106 cfu g-1 at the time of consumption
for Lactobacillus acidophilus, bifidobacteria and other probiotic bacteria
(Boylston et al., 2004).
A prebiotic can be defined as a not digestible food ingredient that beneficially
affects the human body, selectively stimulating the increase and activity
of one or a limited group of colon bacteria. This concept involves certain
dietary compounds resistant to digestive enzyme hydrolysis and/or that
are not absorbed in the superior portion of gastrointestinal tract, including
the small bowel. In fact, these compounds need to get to the large bowel,
where the greater part of the gut microbiota is located and stimulate
the growth of some beneficial microorganisms in the gut (Roberfroid, 2002).
Inulin is an oligosaccharide extracted from commonly consumed plants like
onions, asparagus root, wheat, banana, Chinese chives, garlic, honey,
oat, pine and chicory. It is officially recognized as a natural food ingredient
and is classified as a dietary fibre in most European countries (Roberfroid,
1999). The term synbiotic is used when a product contains both probiotic
and prebiotic ingredients. The synergism is attained in vivo by
the ingestion of lactobacilli on one hand and by the promotion of indigenous
bifidobacteria on the other hand. Thus, a product containing inuline or
oligofructose and probiotic bifidobacteria or L. paracasei, for
example, would fulfil the definition. Synbiotic products have not been
intensively studies to date (Bielecka et al., 2002). Roberfroid
(2002) suggested that these products can improve the survival of bacteria
when they pass into the upper part of the gastrointestinal tract and produce
greater effects in the large bowel. It is not known if the individual
advantages might be additive or even synergistic (Bielecka et al.,
2002).
Aerated dairy desserts have shown a great market potential as a function
of consumer behaviour, interested in lighter and healthier relish products.
Mousse is an aerated dessert with stabilized foamy structure that, although
traditionally home-made, is nowadays produces on an industrial scale and
is gaining space in the dessert market. The most popular mousse flavour
is chocolate, followed by orange, lemon and strawberry. The industrial
production of aerated dairy dessert is delicate, requiring knowledge about
the formation and stabilization of foam, the use of functional ingredients.
The present study was carried out to develop a chocolate mousse to which
probiotic and prebiotic ingredients were added and to verify the perspectives
of the product with regard to potential for consumer health benefits and
to sensorial acceptance.
MATERIALS AND METHODS
Procurement of ingredients and chemicals: The present study was
conducted at Food Biotechnology Laboratory, Sardar Patel University, Vallabh
Vidyanagar during December 2007 to April 2008. The following commercial
ingredients were employed for the production of chocolate mousse: Whole
milk cream (25% fat, Amul, India), Cocoa powder (Cadbury, India), Chocolate
powder (Cadbury, India), Unflavoured gelatin powder (Blue bird, India),
emulsifying agent (SUN-BIRD, India), Sugar (Retail shop, Anand, India),
Skimmed milk powder (Sagar, Amul, India), Double toned milk (Amul, India),
MRS broth (Hi-media). Inulin was supplied by Sigma-Aldrich (Germany).
Bacterial strain and culture condition: Lactobacillus paracasei
subsp. paracasei NCDC 022 was obtained from National Collection
of Dairy Cultures (NCDC), Karnal, India. The culture was maintained by
activating in sterile MRS broth and grown for 24 h at 37°C under aerobic
conditions and for long term storage; strain was kept at-20°C in 15%
glycerol. Prior to use, strain was activated 4 times in 14% sterilized
skim milk.
Preparation and production of chocolate mousse: Three pilot-scale
chocolate mousse products, denoted control (C), probiotic (P) and synbiotic
(S) were produced. The potentially probiotic microorganism L. paracasei
subsp. Paracasei NCDC 22 was added to product P. whereas L.
paracasei subsp. Paracasei plus the prebiotic ingredient inulin
were added to product S and neither the probiotic nor the prebiotic ingredient
were added to product C. Table 1 shows the list of ingredients
and amounts used for Control, Probiotic and Prebiotic chocolate mousse
production. The procedure for preparation of chocolate mousse was followed
according to the method given by Aragon-Alegro et al. (2007). All
ingredients, except the emulsifying agent are mixed well. The mixture
is heated upto 80-85°C in a water bath. Immediately, the mixture is
cooled to 40°C in an ice bath with continuous stirring. To cool the
mixture, the emulsifying agent is added and blended with a hand blender
at 14°C in an ice bath for incorporation of air. The chocolate mousse
was ready to be packaged in covered plastic cup. The packaged mousse was
stored at 4°C for up to 28 days. Each lot of chocolate mousse was
produced in amounts to obtain 800 g of the final product. Chocolate mousse
taken for analysis was the fresh product (day 0) and the stored product
after 4, 7, 14, 21 and 28 days of storage. At each sampling day, microbiological
analysis and physicochemical analysis was carried out apart from sensory
evaluation.
| Table 1: |
List of ingredients and amounts used for chocolate mousse
production |
|
| (Source: Aragon-Alegro et al., 2007) C: control,
P: probiotic, S: synbiotic |
Physico-chemical analysis: The pH values of mousses were determined
with a digital pH meter. Titrable acidity was measured according to the
method of AOAC (1984) and expressed in terms of mL/100 g.
Microbiological analysis: Microbiological analysis was carried
out for all trials of chocolate mousse studied (C, P and S). At each sampling
day, 10 g samples were collected aseptically and blended with 90 mL of
0.1% sterile peptone water and submitted to serial dilutions.
Total plate count, coliform count, E. coli count and yeast and
mould count was carried out according to Bacteriological Analytical Manual
(1998). Nutrient agar media was used for total plate count. Violet Red
Bile Agar (VRBA) was used for the enumeration of coliforms and Eosine
Methylene Blue (EMB) agar was used for the enumeration of E. coli,
whereas enumeration of yeast and moulds were carried out on Potato Dextrose
Agar (PDA). After incubation time, the colonies were counted and the results
were expressed as colony forming units per gram of product (cfu g-1).
L. paracasei count was carried out according to Aragon-Alegro
et al. (2007) on DeMan-Rogosa-Sharpe agar (MRS agar) acidified
to pH 5.4 with acetic acid. Anaerobic incubation was carried out at 37°C
for 3 days. After this time, the colonies were counted and the results
were expressed as colony forming units per gram of product (cfu g-1).
Sensory analysis: Comparison of chocolate mousse samples from
products C, P and S was also conducted by means of sensory evaluation,
employing a Randomized Complete Block Design, using Preference-Ranking
test (Lawless and Heymann, 1999). Sensory evaluation of the mousses was
carried out at the Department of Home Science on 0 day, 4th day and 7th
day of storage by 6 consumers (panelists) of the faculty staff and students,
which were selected based on their interest and chocolate consuming habits.
Samples were presented in white plastic cups and the panel was asked to
evaluate the three-digit coded samples of the three different trials of
mousse(C, P and S all of them prepared on the same day), using a score
from 1 (preferred sample) to 3 (least preferred sample), based on over
all impression. The samples were divided into six blocks, with three repetitions
for each one. This blocking was essential to avoid a tendency to confer
lower or higher grades for samples tasted first.
Acceptability of inulin level in the products: This was performed
to check the acceptable higher concentrations of inulin in chocolate mousse
preparations. Chocolate mousse was prepared with 9, 11, 13 and 15% concentrations
of inulin. Acceptable higher concentrations were determined by means of
Sensory Evaluation, employing a Hedonic Scale Ranking Test. A 9-point
hedonic scale test (1 = extremely dislike, 9 = extremely like) was used
to evaluate sensory attributes for color, appearance, flavor, taste and
overall acceptance. Sensory Evaluation of the freshly prepared mousses
was carried out at the Department of Home Science, at 0 day by 6 consumers
(panelists) of the faculty staff and students.
The physiochemical and microbiological analysis was carried out as previously
mentioned in the study, i.e., at 0 day and on the 7th day as a part of
the storage study.
Statistical analysis: Data was subjected to Analysis of Variance
(ANOVA) using Statistical SPSS for Windows 10.0. Differences between variables
were tested for significance by using a one ways analysis of variance
procedure, Duncan, using a level of significance p≤0.05.
RESULTS AND DISCUSSION
Table 2 shows the mean pH values of the control and
experimental chocolate mousse during storage. pH value of freshly
prepared chocolate mousse was found to be 6.57 for the control (C) and
the two experimental products. Storage period was 0-28 days at
refrigerator temperature. During the storage period there were no significant
difference between control and experimental products of chocolate mousse.
pH values showed a fall from 6.57 on 0 day to 6.40-6.43 on the 14th day
considering all the three products. No significant differences (p≤0.05)
were observed.
From the 14th day onwards, the control product showed a lesser fall in
pH compared to the two experimental products both of which registered
a greater fall. On the 28th day, control showed a slightly higher pH compared
to the probiotic and synbiotic products both of which showed nearly similar
values. Since both the experimental products had probiotic microorganisms
L. paracasei and their activity has led to a drop in the pH
which is not seen in the control product. On 0, 4th and 7th day, between
the three products no significant difference (p≤0.05) was observed.
According to Beresford et al. (2001), the optimal pH for the growth
of the most common bacteria is near to neutral and this growth is suppressed
in pH values below 5.0. Thus the decrease in pH values in all
the three products was not sufficient to impair the survival of probiotic
micro-organisms present in the chocolate mousse.
Table 3 shows the mean titrable acidity values evaluated
during the storage of the control and experimental chocolate mousse products.
Titrable acidity showed a value of 0.27 on 0 day for all the three products.
The titrable acidity did not show any change in value for any of the three
products until the 7th day, indicating that the chocolate mousse, whether
control or experimental, retained it`s freshness for a minimum period
of seven days without any difficulty. Thus no significant differences
(p≤0.05) were observed between the three products or for each product
as days of storage increased up to the 7th day.
Comparing the titrable acidity from the 14th day onwards, there was a
significant rise in titrable acidity in each product as the days of storage
increased. Similarly the two experimental products showed slightly higher
acidity compared to the control from the 14th day onwards. On the 28th
day again both experimental products showed significantly higher (p≤0.05)
titrable acidity compared to control. It is seen that titratable acidity
of the products increased during 28 days of the storage period, probably
due to the presence of L. paracasei (Aragon-Alegro et al.,
2007).
| Table 2: |
pH values of control, probiotic and synbiotic
chocolate mousse products during 28 days of storage period |
|
Mean of three trials±SEM. Values carrying
the same superscript are not different from each other. * indicates
significant difference at p≤0.05, C: Control; P: Probiotic; S:
Synbiotic |
| Table 3: |
Titratable acidity of control, probiotic and synbiotic
chocolate mousse products during 28 days of storage |
|
| Mean of three trials±SEM; Values carrying the
same superscript are not different from each other; * Indicates significant
difference at p≤0.05; C: Control; P: Probiotic; S: Synbiotic |
Table 4 shows the mean values of total count and viability
of L. paracasei in the control and two experi-mental products.
For total count, there was no growth on 0 day for control, while the experimental
products showed values of 5.05x107 and 4.95x107
cfu g-1, respectively for P and S, on 0 day. Control product
showed zero count of bacteria until the 7th day. The total count did not
show much change in values for both the experimental products until the
7th day, indicating that the experimental chocolate mousse retained it`s
freshness for a minimum period of seven days without any difficulty, thus
no significant differences (p≤0.05) were observed between the two experimental
products as days of storage increased up to the 7th day. Comparing the
total count from the 14th day onwards, there was a significant rise in
total count as the days of storage increased for both the experimental
products, although the counts did not show any significant difference
on the 14th day. Significant differences were observed between the two
experimental products, the inulin added synbiotic product showing higher
values from the 21st day onwards, indicating the beneficial synbiotic
effect from the 21st day onwards.
Meanwhile, from the 14th day onwards the control also showed the appearance
of bacteria, as per the total count results. The control values were lower
than the two experimental products, on the 14th, 21st and 28th day, showing
significant differences for the 21st day and 28th day.
| Table 4: |
Microbiological counts of Total count and L. paracasei
of control and experimental chocolate mousse products |
|
| Mean of three trials±SEM; Values carrying the
same superscript are not different from each other; * Indicates significant
difference at p≤0.05; C: Control; P: Probiotic; S: Synbiotic; results
expressed in cfu/g |
L. paracasei count also showed a similar trend as that for total
count. The control product showed zero count of L. paracasei as
expected from 0 day to the 28th day. In the two experimental products
the counts showed an increasing trend as the days of storage progressed.
In the probiotic product, a significant increase in the count was observed
on the 28th day compared to the previous days of storage. In the synbiotic
product, the L. paracasei count was slightly low on 0 day which
continued to show an increase (none significantly) as the day of storage
increased. A significant increase was noted from the 21st day onwards.
Comparing the counts in both the experimental products it is seen that
the synbiotic product showed higher counts indicating that inulin promoted
an increase in the count of L. paracasei. Between the two products
a significant difference (p≤0.05) was observed on the 21st and 28th
day. Administration of inulin to human volunteers on a defined diet has
been reported to result in increased numbers of probiotic bifidobacteria
and reduced bacteroides, clostridial and fusobacterial populations in
feces (Gibson et al., 1995; Kleesen et al., 1997).
According to Boylston et al. (2004), the recommended levels of
probiotic microorganisms in food at the time of consumption is 106
cfu g-1, to have a beneficial effect on consumer health. This
criterion is fulfilled in the chocolate mousse developed in the present
study as a potential vehicle for L. paracasei.
Aragon-Alegro et al. (2007) monitored chocolate mousse products
similar to the present study that is control, probiotic and synbiotic
products for 28 days to assess the population of the probiotic L.
paracasei as well as contaminants, during storage at 4°C and reported
that the probiotic was still viable after 28 days, maintaining population
levels about seven log cfu per gram and reported that L. paracasei
increased slightly in probiotic chocolate mousses during 21 days of storage.
Similar result was found in our study. Total count and L. paracasei
count increased in the probiotic as well as in the synbiotic chocolate
mousse product during 14 days of storage. Vinderola et al. (2002)
evaluated the suitability of Argeatinean fresco cheese as a food carrier
for probiotic cultures. Their study demonstrated that this cheese may
used as a vehicle for probiotic bacteria. The cultures bifidobacteria,
L. acidophilus and L. casei survived satisfactorily until
the 60th day. In the present study, viability of L. paracasei increased
from 3.9x107 to 1.6x109 cfu g-1 on the
28th day in the probiotic chocolate mousse during 21 days of storage at
refrigerator temperature.
Table 5 shows the population of the contaminants namely
coliforms, E. coli and Yeast and Mold during the storage period
for the different products of chocolate mousse. Coliforms and E. coli
were detected only after 21 days product C, values were 3x103 and
1x103 cfu g-1, respectively.
Upto 21 days of storage both coliform and E. coli were not detected
in the experimental products P and S. On the 28th day of storage for coliforms
and E. coli in product P; values were 1.5x104 and 1x104
cfu g-1, respectively; and 2.0x104 and 6x103
cfu g-1 for coliform and E. coli for respectively, for
product S. Yeasts and mold were undetected throughout the storage period
for all the three products.
Coliforms and E. coli count for all the three products indicates
that the product has been prepared with high quality raw ingredients and
that almost care has been taken during the preparation of mousse. Further
all the three products show a high shelf life upto 28 days of storage.
Aragon-Alegro et al. (2007) noted that the growth of yeasts and
moulds might limit the shelf life of the developed product chocolate mousse.
In their study growth was detected after 14 days for both the control
and probiotic mousses. However, growth for the synbiotic mousse was not
observed until 21 days of storage in their research, whereas in our study
the results showed that coliform and E. coli were not detected
until 28 days of storage in both the experimental products, while Yeast
and Mold were not detected at all during the storage study.
Table 6 shows the mean sensory evaluation scores evaluated
for the fresh and stored, control and experimental chocolate mousse samples.
Sensory scores indicated no significant differences in preference between
fresh samples (0 day) of mousse even though the synbiotic product was
considered the most preferred product of chocolate mousse on the basis
of a slightly higher score. The scores on the storage of the products
indicated a decline for both control and probiotic products and an increase
in acceptability for the synbiotic product, although no significant differences
(p≤0.05) were observed. The results indicate that compared to the control
and the probiotic product, synbiotic product showed higher acceptability
as days of storage increased.
| Table 5: |
Microbiological counts of contaminant population of
control and experimental chocolate mousse products |
|
| Mean of three trials±SEM; Values carrying the
same superscript are not different from each other; * Indicates significant
difference at p≤0.05; C: Control; P: Probiotic; S: Synbiotic; results
expressed in cfu g-1 |
| Table 6: |
Sensory evaluation of fresh and stored control and experimental
chocolate mousse products |
|
| Mean of six panelists±SEM; Scale of preference
ranged from 1 to 3 (1-most liked; 2-moderately liked; 3-least liked);
Values carrying the same superscript are not different from each other;
*Indicates no significant difference; C-control; P-probiotic; S-synbiotic |
| Table 7: |
Sensory evaluation synbiotic chocolate mousse products
prepared incorporating inulin at higher levels |
|
| Mean of six panelists±SEM; Scale of preference
ranged from 1 to 9, (1-like extremely to 9 dislike extremely); Values
carrying the same superscript are not significantly different from
each other; * Indicates significant difference at p≤0.05 |
Acceptability of inulin level: Table 7 shows
the mean sensory scores for freshly prepared synbiotic chocolate mousse
incorporating inulin at higher levels. The results showed no significant
differences between the four experimental products namely B, C, D and
E which showed a range from 3.25 to 4.17. Product B (9% inulin) was more
acceptable with a mean score of 3.25 than the other synbioitc products
but product C (11% inulin) also showed nearer scores i.e., 3.75. Product
A product without inulin showed significantly higher acceptability. Again,
increasing the inulin levels to 13% decreased the acceptability.
CONCLUSION
The present study showed successful incorporation of L. paracasei
and inulin in chocolate mousse. Inulin did not interfere with the viability
of L. paracasei. The present investigation can be further expanded
by using different probiotic and prebiotic sources like probiotic strains
L. burgaricus, L. casei, L. acidophilus or bifidobacterim
and for prebiotic source soya-oligofructosaccharide, galacto-oligosaccharides,
xylo-oligosaccharides may be used.
