Abstract: Eight commercial probiotic fermented milk products (six full fat and two low fat) from Al-Ahsa markets were evaluated for chemical, microbiological and sensory properties. The chemical composition parameters ranged from 0.9-1.2% fat (low fat products), 3.0-3.9% fat (full fat products), 3.1-4.7% protein, 0.7-1.2% ash and 7.5-3.7% carbohydrate in all the milk products. The pH values of all the products decreased significantly from the production day to the end of storage period. With respect to the microbiological side, the coliform bacteria, moulds and yeasts counts were not detected in all the products during the refrigerated storage at 5 ±1 °C. However, seven out of eight products contained over 106 cfu mL-1 of bifidobacteria on the production day. Only two of these products maintained 106 cfu mL-1 viable count of bifidobacteria till the end of cold storage period. On the other hand, three out of eight products showed the highest number of L. acidophilus viable count (above 108 cfu mL-1) on production day. The results of sensory evaluation showed that all the tested products obtained high scores for flavor, appearance, texture or consistency and smell (odor) properties during the storage period. These results suggest that for optimum benefits, the probiotic fermented milk products with live probiotic bacteria should be consumed within one week of their production date. The research provided useful information to the dairy industries to develop new technology to ensure the supply of high quality milk products to the consumers.
INTRODUCTION
Generally, the fermented dairy products are considered safe and nutritious. The beneficial effects of fermented milk products may be further enhanced by supplementation of probiotic bacteria such as Lactobacillus and Bifidobacterium species. A probiotic is generally defined as a live microbial supplement which beneficially affects the host animal by improving its intestinal microbial balance (Fuller, 1989). Several health benefits are related to the regular consumption of viable probiotic bacteria including improvement of lactose tolerance (Kim and Gilliland, 1983) antimicrobial (Yildirim and Johnson, 1998), anti-carcinogenic (Abd El-Gawada et al., 2004), hypocholesterolemic (Kikuchi-Hayakawa et al., 2000; Abd El-Gawada et al., 2005) and anti-mutagenic (Hsieh and Chou, 2006). Fermented dairy products are considered as vehicles by which the consumers might receive adequate numbers of probiotic bacteria (Samona and Robinson, 1994; Stanton et al., 1998). In order to produce therapeutic benefits, sufficient number of viable microorganisms must be present throughout the entire shelf life of the product. In this regard, the ranges of minimum levels for probiotic bacteria in fermented milks were suggested from 105-106 cfu mL-1 (Samona and Robinson, 1994). Schuller-Malyoth et al. (1968) reported that a good probiotic culture should contain between 106 and 108 viable cells per milliliter. For bifidobacteria to provide therapeutic benefits, it was recommended that they must be viable and ingested in numbers ≥ 106 cells per gram of yoghurt (Kurman and Rasic, 1991). Therefore, maintaining the viability of probiotic bacteria until the products are consumed is of great interest for the dairy industries. Several factors such as acidity, pH, hydrogen peroxide, oxygen content, temperature of storage at the time of production and the storage facilities of fermented milk affect the viability of probiotic bacteria in yoghurt (Samona and Robinson, 1994; Lankaputhra and Shah, 1995; Lankaputhra et al., 1996). Recently, a wide variety of probiotic fermented milk products are commercially available in the Saudi Arabia. These products are exposed to high temperature during handling and transportation and to cold storage in the markets for minimum one week until consumption in Saudi Arabia. The aim of this study was to determine the viable count of probiotic bacteria (Lactobacillus acidophilus and bifidobacteria) in some probiotic fermented milk products from Al-Ahsa markets during refrigerated storage and also to assess the effect of pH and cold storage period at 5± 1 °C on the viability of probiotic bacteria in these milk products.
MATERIALS AND METHODS
Collection of Fermented Milk Products
Eight commercial probiotic fermented milk products (six full fat and
two low fat) were collected on production day from Al-Ahsa markets, Saudi
Arabia during 2007. These samples were analyzed microbiologically on the
production day and then every week for three weeks during refrigerated
storage at 5± 1 °C. The same samples were stored in the refrigerator
for chemical analysis. A brief description of these products and their
labeled ingredients are shown in Table 1.
Analytical Methods
Chemical Composition
Moisture contents of probiotic fermented milk products was determined according
to the Association of Official Analytical Chemists method (AOAC,
1995). Total Nitrogen (TN) was determined by Kjeldahl Method (AOAC,
1995) and fat content by the Gerber method as described in the British
Standard method (1989). The ash content was determined according to the
AOAC method (AOAC, 1995).
Total carbohydrate was calculated by difference as follows (Manzi et al., 2007):
Carbohydrate (g/100 g) = 100 – (Water+Protein+Fat+Ash) |
Total energy was calculated according to the following equation (CEE Directive, 1990):
Energy (kcal/100 g) = 4x(g protein+g carbohydrate)+9x(g
lipid) |
| Table 1: | Description of the commercial probiotic fermented milk products |
pH Determination
The pH of probiotic fermented milk products was measured with a digital
pH meter by using a TPS digital pH meter (Denver Instruments, TX, USA).
The pH of yoghurt samples was determined by direct immersion of the electrode
into the sample (20-25 mL) at room temperature.
Coliform Bacteria
The count of coliform group was estimated by plating on McConkey agar medium
(Oxid), as recommended by the APHA (1992). The plats were
incubated at 37 °C for 2 days.
Moulds and Yeasts
Potato dextrose agar medium (Oxid) was used for enumerating yeasts and moulds
count according to APHA (1992). The plats were incubated
at 20-25 °C for 2-3 days.
Bifidobacteria Estimation
The count of bifidobacteria was enumerated according to the method of Dinakar
and Mistry (1994), in which a mixture of antibiotics, including 2 g paromomycin
sulphate, 0.3 g nalidixic acid and 60 g lithium chloride, was dissolved in 1
L distilled water, filter-sterilized (0.2 μm) and stored at 4 °C until use.
The antibiotic mixture (5 mL) was added to 100 mL MRS-agar medium. L-Cysteine-HCl
0.5% (w/v) (Sigma Chemical Co., St. Louis, MO, USA) was also added to decrease
the redox potential of the medium. Plates were incubated at 37 °C for 48 h
an-aerobically.
Lactobacillus acidophilus
The count of Lactobacillus acidophilus was determined according
to Van de Casteele et al. (2006) by using MRS
medium + 0.5 ppm filter sterilized clindamycin. Plates were incubated at 37
°C for 48 h an-aerobically.
Sensory Evaluation
Sensory evaluation of commercial probiotic fermented milk products was carried
out on the production day and for 3 weeks of cold storage using a regular score
panel according to Tamime and Robinson (1985).
RESULTS AND DISCUSSION
Chemical Composition
The results showed great variability in the chemical composition of different
fermented milk products (Table 2). The ranges of various chemical
components were 0.9-3.9% fat, 3.1-4.7% protein, 0.7-1.2% ash and 7.5-3.7% carbohydrate.
Low fat products such as D and F were high in water content and less total energy
than other products. However, product A showed high protein and carbohydrate
contents and less moisture content than other tested products thus showing more
energy. Whereas, the fat contents of B and E products were higher than other
products (3.9%). Whereas, G, A and D products were higher in ash contents when
compared with the other products. In Italian market, Manzi
et al. (2007) reported that the fat, protein and ash content of probiotic
fermented milk ranged from 0.2-3.6, 2.7-5.8 and 0.4-0.8%, respectively.
| Table 2: | Chemical composition (g/100 g) of commercial probiotic fermented milk productsa |
aAnalytical data are means of triplicate
analysis standard deviation, bProduct samples as in Table
1 |
|
| Table 3: | Changes in pH values of probiotic fermented milk products during refrigerated storage period 5± 1 °C |
a-dMean values (± SD; n = 3) in row
with the same letter(s) are not significantly different from each
other at p>0.05, *Product samples as in Table 1,
ψProduct in one-day old |
|
Changes in pH Values
The initial pH values of fermented products ranged from 4.43-4.76. In general,
the pH of all the tested products decreased gradually from the production day
to the end of storage period (Table 3). The difference in
pH was significant between the production day and the end of storage period.
There were no significant differences in pH values among A, B and C products
between the production day and the first week of storage. However, H product
showed significant decrease in pH (3.30) which could be due to the presence
of three strains of starter Lactobacillus helveticus, Bifidobacteria
and Lactobacillus acidophilus (Table 1) compared with
other products. Shah et al. (1995) also found
similar decreases in pH values during storage of commercial yoghurts containing
L. acidophilus and B. bifidum. Similarly, the initial pH values
in yoghurts containing L. acidophilus and bifidobacteria decreased from
4.33-4.41 at day 0 to 4.16-4.22 at the end of 35 days of storage (Dave
and Shah, 1997). In parallel, Akalin et al. (2004)
found the initial pH values of different types of yoghurt in the range of 4.51-4.48
which slightly decreased during storage.
Coliform Bacteria, Moulds and Yeasts
The coliform bacteria and moulds and yeasts counts were not detected
in all tested products on the production day and during the refrigerated
storage at 5± 1 °C for 3 weeks. This could be attributed to
the high hygienic systems implemented in these factories.
Survival of Bifidobacteria
The populations of bifidobacteria decreased significantly from the production
day to the end of refrigerated storage period at 5± 1 °C in all the commercial
probiotic fermented milk products (Table 4). However, during
the two weeks of cold storage, the population of bifidobacteria remained above
105 cfu mL-1. The viability losses of bifidobacteria showed
between 0.5 and 3.74 log cycles in F and D products, respectively. Interestingly,
the G product recorded maximum viable count of bifidobacteria on the production
day as well as at the end of storage (9.88 and 6.71 log cfu mL-1,
respectively) than all the other products. This could be due to the highest
pH value of G product than other products (Table 3). Whereas,
F product showed minimum count of this organism. According to Kurman
and Rasic (1991), the viable level of bifidobacteria must be above 106
cfu mL-1 to provide therapeutic benefits. Among the various products,
B and G fulfilled this requirement for viability of bifidobacteria till the
end of cold storage period. The reduction of bifidobacterial count may be due
to the decrease of pH values, post process acid production (Wang et al.,
2002), sensitivity to oxygen (Shimamura et al., 1992),
metabolites such as hydrogen peroxide and ethanol and to bacteriocins produced
by lactic acid bacteria (Frank and Marth, 1988). These
results were in agreement with those of (Samona and Robinson,
1994; Medina and Jordano, 1994; Lankaputhra
and Shah, 1995; Lankaputhra et al., 1996),
who found poor viability of bifidobacteria in yoghurt during storage.
| Table 5: | Survival (log cfu mL-1) of bifidobacteria in commercial
probiotic fermented milk products during refrigerated storage |
a-dMean values (± SD; n = 3) in row with the
same letter(s) are not significantly different from each other at p>0.05,
*Product samples as in Table 1, ψProduct
in one-day old |
|
| Table 5: | Survival (log cfu mL-1) of Lactobacillus acidophilus in commercial probiotic fermented milk products during refrigerated storage |
a-dMean values (± SD; n = 3) in row
with the same letter(s) are not significantly different from each
other at p>0.05, *Product samples as in Table 1,
ψProduct in one-day old |
|
Survival of Lactobacillus acidophilus
Generally, the viability of L. acidophilus decreased gradually
and significantly in commercial probiotic fermented milk products during cold
storage (Table 5). Among all the products, A, B and G recorded
the highest number of L. acidophilus viable count (8.66, 8.66 and 9.53
log cfu mL-1, respectively) on the production day. On the other hand,
the C, D and F products showed minimum count of this organism. During storage
period, the viable count of the bacteria ranged between 3.7 and 7.5 log cfu
mL-1 in all the products. Speck (1976) reported
that 108 to 109 viable cells of L. acidophilus
should be ingested daily to ensure that consumers receive health benefits. Among
all the products A, B and G fulfilled this requirement for viability of L.
acidophilus on the production day. The reduction in the count of L. acidophilus
during the cold storage may be due to the production of antimicrobials such
as bacteriocins, H2O2, or organic acids. Characterizing
bacteriocins and bacteriocin-like inhibitory substances produced by L. acidophilus
and other lactic-acid bacteria were reported by Shah and
Dave (2002). These results agree with those of Shah et
al. (1995), who reported that three out of five brands of fresh yogurt
contained 107 to 108 viable cells of L. acidophilus
per gram while the remaining two brands contained less than 105 L.
acidophilus cells per gram. Nighswonger et al.
(1996) found that some strains of L. acidophilus lost viability during
storage at 7 °C after 28 days. Dave and Shah (1997)
found that the survival of L. acidophilus in yogurts after 35 days of
storage was approximately 0.1 to 5% as compared to after 5 days of storage.
Olson and Aryana (2008) found that the L. acidophilus
counts in yogurt tended to decrease from 6.84 to 4.43 log cycles during 8 week
of storage time.
| Table 6: | Sensory evaluation of commercial probiotic fermented milk products during storage period |
a-dMean values (± SD; n = 3) in row
with the same letter(s) are not significantly different from each
other at p>0.05, *Product samples as in Table 1,
ψProduct in one-day old |
|
Sensory Evaluation
All the tested products scored high points in flavor, appearance,
texture or consistency and smell (odor) during the refrigerated storage
period (Table 6). However, there was no significant
difference (p>0.05) in the appearance, body and texture and smell (odor)
of all tested products during storage period. With respect to the flavor
score, the A, B, E and G products showed significant differences in the
3rd week of storage period.
Formation of exopolysaccharide by the starter and probiotic cultures may contribute to prevention of synersis and an increase in viscosity, combined with a better mouth feel (Hussein et al., 1996). Griffin et al. (1996) reported that polysaccharide producing yoghurt bacteria were important determinants of yoghurt viscosity and texture. These starter cultures improve the viscosity of yoghurt leading to resistance to mechanical damage (Tamime and Deeth, 1980).
CONCLUSIONS
Seven out of eight products contained over 106 cfu mL-1 of bifidobacteria on the production day. Only two of these products maintained 106 cfu mL-1 viable count of bifidobacteria till the end of cold storage period. Three out of eight products showed the highest number of L. acidophilus viable count (above 108 cfu mL-1) on production day. The sensory evaluation showed that all the tested products obtained high scores for flavor, appearance, texture or consistency and smell (odor) properties during the storage period. These results suggest that to obtain optimum health benefits, the probiotic fermented milk products with live probiotic bacteria should be consumed within one week of their production date. The research provided useful information to the dairy industries to develop new technology to ensure the supply of high quality milk products to the consumers.