Consumers across the world are becoming more interested in foods with health promoting features as they gain more awareness of the links between food and health. Among the functional foods, products containing probiotics are showing promising trends worldwide.
Probiotics such as Lactobacillus and Bifidobacterium sp. are
bacterial members of the normal human intestinal flora that exert several beneficial
effects on human health and improve the intestinal microbial balance, resulting
in the inhibition of bacterial pathogens, reduction of colon cancer risk, improving
the immune system and lowering serum cholesterol levels (Tamime
et al., 2005; Saarela et al., 2002;
Isolauri et al., 2001).
Recently, the food biotechnology industry has developed a number of commercial products containing a single probiotic strain or bacterial associations of various complexities.
Also, Lactic Acid Bacteria (LAB) and its metabolites have shown to play an
important role in improving microbiological quality and shelf-life of many fermented
food products. Dairy products have long been consumed by consumers and provide
a good example of bio-preservation (Zottola et al.,
Today LAB is a focus of intensive international research for its pivotal role in most fermented foods.
Furthermore, LAB strains synthesize short chain fatty acids, vitamins, and
exopolysaccharides (EPS) that are employed in the manufacturing of fermented
milk to improve its texture and viscosity (Curk et al.,
Development of dairy products with new products and flavors has potential health
benefits thereby increasing sales and consumers satisfaction.
|| Denak (Oliveria decumbens Vent.)
Traditional preparation of milk and yoghurt may be beneficial by including
other ingredients such as soya protein, vegetables, sweet potato, pumpkin and
plum to enhance the flavor as well as the nutritional quality (Joo
et al., 2001).
However, traditional medicinal plants such as Denak (Oliveria decumbens
Vent) has been proved to provide important therapeutic values. The plant
Oliveria decumbens Vent. (Apiaceae), with the common Persian names of
Mooshkorok, Den, and Denak, is found in south
and south west parts of Iran and is endemic to Iran. Denak (Oliveria decumbens
Vent.) belongs to Umbelliferae family and is an endemic plant of Flora Iranica
that grows in high temperature areas of south and west of Iran (Fig.
1) (Mozaffarian, 1996).
Denak (Oliveria decumbens Vent.) is a relatively less explored plant.
The limited researches about therapeutic property of this plant are reported
antibacterial and antifungal activity of its essential oil. Recently the study
of researchers showed that Denak (Oliveria decumbens Vent.) has proper
antibacterial effect and can be considered as a new source of antibiotic discovery
and development for infection disease treatment purpose (Amin
et al., 2005).
In the present study, we investigate first, the ability of Lactobacillus
acidophilus and Bifidobacterium bifidum to grow and survive in presence
of Denak (Oliveria decumbens Vent.) during 21 day of refrigerated storage
and evaluate the organoleptic properties of milk and yoghurt.
MATERIALS AND METHODS
Sample collection and preparation: The plant materials of O. decumbens Vent was collected from Kazerun located in Fars province, in the south west of Iran, in July 2012. The flowers of O. decumbens Vent were dried under shade at room temperature for 17 days. Low-fat sterilized milk and yoghurt (1.5%) were locally purchased (Kazerun, Iran). Commercially available probiotic cultures of Lactobacillus acidophilus LAFTI® L10 and Bifidobacterium bifidum LAFTI® B94 were obtained from DSM Food Specialities Australia Pty Ltd. (Moorebank, NSW, Australia). MRS Agar culture medium was used for carrying out the microbial test (MERCK, Germany).
Preparation of probiotic Bifidobacterium bifidum milk containing Denak powder (Oliveria decumbens Vent) powder at first passage: In order to produce milk containing the probiotic bacterium Bifidobacterium bifidum, four containers each containing 1 L of low-fat sterilized milk (1.5% fat) were considered as our four groups. The starter (Bifidobacterium bifidum) was added directly to all the containers, followed by adding Denak powder of 0 (Control sample), 0.5, 1 and 1.5% to all the containers, respectively and finally they were placed in the incubator at 38°C. The acidity test was performed approximately every 2 h until reaching 42° Dornic. The samples were then taken out of incubator and transferred to a refrigerator and stored at 2°C. The produced probiotic milk was evaluated once every 7 days by counting the microbes using direct counting method.
Preparation of probiotic Bifidobacterium bifidum yoghurt containing Denak powder (Oliveria decumbens Vent) at the second passage: To produce Biidobacterium bifidum yoghurt in this stage, after providing 4 containers, 1 L of the low-fat sterilized probiotic milk (1.5 % fat) from the control group at first passage and the (1.5%) starter of low-fat yoghurt (1.5%) were added to each container. Different concentrations of Denak powder (0, 0.5, 1, and 1.5%) were added, respectively to the containers and mixed properly so that Denak powder was uniformly dissolved. Afterwards, all the containers were placed in the incubator at 38°C. Approximately every 2 h, the acidity and pH tests were done until acidity reached 90° Dornic. Then, the samples were taken out of the incubator and transferred to a refrigerator and stored at 2°C. The produced probiotic Denak yoghurt was evaluated every 7 days by counting the microbes using direct counting method and after 10 days the yoghurt was evaluated for sensory properties, using questionnaires filled by 15 participants. The respondents were asked to rate the factors of scent, taste and permanence on a scale ranging from very good, good, medium, to weak. The results were analyzed in a statistical descriptive test by SPSS version 17 software.
Preparation of probiotic Lactobacillus acidophilus milk containing Denak powder (Oliveria decumbens Vent) at first passage: All the same procedures were followed as mentioned above with the difference of using Lactobacillus acidophilus instead of Bifidobacterium bifidum.
Preparation of probiotic Lactobacillus acidophilus yoghurt containing Denak powder (Oliveria decumbens Vent) at second passage: All the same procedures were followed as mentioned above with the difference of using Lactobacillus acidophilus instead of Bifidobacterium bifidum.
Having produced the above-mentioned products, we stored 1000 g of each product in a disposable container placed in a refrigerator for 21 days. During this period, each sample was tested in days 1, 7, 14 and 21 for acidity, pH and sensory properties.
Statistical analysis: All the above experiments were repeated three times with each test carried out in triplicate. SPSS17 was used for one-way analysis of variance for all data, and significant differences (p<0.05) among means were determined by the least significant difference test.
Table 1 and 4 show the acidity degrees
of Denak milk and yoghurt in Lactobacillus acidophilus and Bifidobacterium
bifidum samples during storage time in the refrigerator. The results of
these tables show the positive correlation between increased acidity value and
increased Denak concentration which the samples containing 1.5% Denak powder
in milk and yoghurt had high acidity value than the other samples investigated.
||The acidity level based on Dornic degree in the Denak Lactobacillus
acidophilus milk and yoghurt within 21 day storage in the refrigerator
||Growth of microbes in the Denak Lactobacillus acidophilus
milk and yoghurt
||Microbial growth on MRS-A cultivation environment of Lactobacillus
acidophilus Denak milk and yoghurt at refrigerator during 21 days insolubility
||Acidity level based on Dornic degree in the Denak Bifidobacterium
bifidum milk and yoghurt within 21-day storage in the refrigerator
||Growth of microbes in the Denak Bifidobacterium bifidum
milk and yoghurt
Table 2 and Table 5 show the growth rates of microbes in Denak milk and yoghurt in Lactobacillus acidophilus and Bifidobacterium bifidum samples at storage time. The results show that the growth rate of bacteria was increased by increasing the concentration of Denak powder and reached the desired acidity at shorter period.
Table 3 shows the microbial growth on MRS-A cultivation environment of Lactobacillus acidophilus Denak milk and yoghurt at refrigerator during 21 day of storage. The samples containing 1.5% Denak powder possessed the highest count of bacteria.
The microbial growth on MRS-A cultivation environment of Bifidobacterium
bifidum Denak milk and yoghurt at refrigerator during 21 days was poor because
Bifidobacterium bifidum has good growth on MRS Broth. The microbial growth
of Bifidobacterium bifidum on MRS Broth was high. It was observed that
Bifidobacterium bifidum has high inhibitory activity in MRS Agar during
21 days of storage. These results showed that Denak was suitable for this intestinal
bacterium that was kept viable up to the end of fermentation (21 days). All
tested Bifidobacterium bifidum was capable of growing well on Denak milk
and yoghurt without nutrient supplementation.
In the present study, the effects of Denak (Oliveria decumbens Vent)
on the growth and viability of the bacteria Bifidobacterium bifidum
and Lactobacillus acidophilus in probiotic milk and yoghurt were investigated.
The acidity, pH and survival of the bacteria in Denak probiotic milk and yoghurt
were evaluated at 2 h intervals till reaching 42° Dornic acidity degrees
for milk and 90° Dornic degree for yoghurt in the incubator at 38°C.
At the first hours of production, the Lactobacillus acidophilus milk
containing 1 and 1.5% Denak powder reached the acidity of 42° Dornic earliest,
followed by 0.5 and 0% milk. Once they reached this acidity level, they were
transferred to a refrigerator at 2°C. The storage time in the refrigerator
was determined to be 21 days. In direct microbial counting in first day, the
highest counts were sequentially in the samples with 0.5, 1 and 1.5% and the
controls, indicating the positive correlation between increased bacterial growth
and increased Denak concentration. Upon evaluation of the cultured samples on
MRS agar media, the same correlation was revealed. The Lactobacillus acidophilus
yoghurt with 1.5% Denak powder reached the acidity of 90° Dornic earliest,
followed by the samples with 1 and 0.5% and the control, Once they reached this
acidity level, they were transferred to a refrigerator at 2°C. The storage
time in the refrigerator was found to be 21 days. Although the basic feature
of the probiotic products consumption is their medicinal effects (bio value),
their associated sensory properties are also important. In other words, sensory
properties rather than medicinal effects play the most important role in their
daily consumptions. Among the probiotic products, fermented ones especially
the probiotic yoghurt is popular worldwide for its unique sensory properties
(Mortazavian and Sohrabvandi, 2006).
The sensory evaluation was performed by 15 participants for the probiotic Lactobacillus acidophilus yoghurt with varying concentrations of Denak powder, after seven days. There were significant differences between the samples (p>0.05) and it was shown that the increase of Denak powder gives rise to favorable taste, color, scent and thickness.
The minimum required level of probiotic bacteria to be useful for the consumers
body is 107CFU mL-1 of living bacteria and the level in
the present study was found to be 1010, thus, it could be beneficial
for the consumers (Marhamatizadeh et al., 2009).
Upon evaluation of the samples on MRS Agar, the Lactobacillus acidophilus with Denak powder had the counts equal to logarithmic 109 in day 14 and the sample product with 1.5% Denak powder possessed the highest count of bacteria.
Bifidobacterium bifidum milk containing 1.5 and 1% Denak powder reached 42° Dornic acidity earliest than others, followed by the milk with 0.5% and finally the control. Once reached 42° Dornic, the samples were transferred to a refrigerator at 2°C. The permanence of the product in the refrigerator was determined to be 21 days during which the acidity of control sample was lower than other samples.
As revealed in direct microbial counting, the count in day 7 was higher, compared to day 1, for all Denak concentrations, but possessed logarithmic coefficient 1010. The bactericidal and inhibitory effect of low pH was stronger for Bifidobacterium bifidum than Lactobacillus acidophilus and it seems that during the storage time and enhanced fermentations process, decreased pH caused decreased growth of Bifidobacterium bifidum.
At the first hours of production, the Bifidobacterium bifidum yoghurt with 1.5 and 1% Denak powder reached 90° Dornic acidity earliest, followed by the yoghurt sample with 0.5% and the control. They were transferred to a refrigerator at 2°C, once reached the 90° Dornic acidity.
The product permanence in the refrigerator was found to be 21 days. No significant difference was observed in the Bifidobacterium bifidum yoghurt with Denak powder in terms of color, thickness, taste and scent. The sample with 1.5% was with the highest bacterial counts, as revealed in the evaluation of the samples in direct counting method.
The results of the studies addressing the probiotic bacteria have demonstrated
the following: The increased concentration of malt and soya caused increase
in the microorganism growth and rising acidity level which in turn resulted
in shorter incubation time for the desired acidity. In a study on the effects
of soya powder on the growth of the bacteria, Lactobacillus acidophilus
and Bifidobacterium bifidum, in probiotic products, it was demonstrated
that the shelf life for the acidity reaching the desired level during incubation
decreased for the milk with both bacteria and combined soya and malt, compared
to the milk with only soya. As for the yoghurt with both bacteria, the same
results were yielded and incubation time for the yoghurt with malt and soya
was decreased (Marhamatizadeh et al., 2009;
The effect of honey on the growth of the above-mentioned bacteria introduced
simultaneously into dairy products and drinks was investigated and the results
indicated that yoghurt with only Lactobacillus acidophilus tasted sourer
than the yoghurt with both bacteria. The products containing Bifidobacterium
bifidum, compared to those with Lactobacillus acidophilus, were with
slower growth rate and also tasted less sour and were of longer permanence.
They were not of favorable taste when honey concentration increased and the
control was of the best taste among all the samples (Marhamatizadeh
et al., 2010).
In another study addressing the effect of cinnamon on the bacterial growth,
it was demonstrated that the increased cinnamon concentration promoted the growth
of the bacteria in probiotic milk and yoghur. (Yaghtin, 2010).
In another study addressing that investigated the effect of spearmint on the
bacterial growth, it was demonstrated that increased spearmint concentration
promoted the growth of Lactobacillus acidophilus and Bifidobacterium
bifidum in probiotic milk and yoghurt (Marhamatizadeh
et al., 2011b).
In another study addressing the effect of juice on the bacterial growth, it
was demonstrated that the increased juice product promoted the growth of the
bacteria in probiotic orange and apple (Marhamatizadeh
et al., 2012a).
In a study that investigated the effect of garlic on bacterial growth and survival,
it was observed that increased garlic concentration promoted the growth and
viability of probiotic bacteria in milk and yoghurt during refrigerated storage
(Marhamatizadeh et al., 2012b).
In another investigation addressing the effect of dill extract on growth and
survival of Lactobacillus acidophilus and Bifidobacterium bifidum,
it was represented that dill extract has positive effect on growth and viability
of probiotic bacteria in milk and yoghurt during permanence period and finally
leaded to produce new fermented dairy product (Marhamatizadeh
et al., 2012c).
In experiment researchers investigated the effect of permeate on the growth
and survival of the above mentioned bacteria (Lactobacillus acidophilus
and Bifidobacterium bifidum) was indicated that the permeate was suitable
support for intestinal bacteria that had kept viable up during 21 days of refrigerated
storage and final evaluation of products showed that permeate can be successfully
used in the preparation of nutritive probiotic beverages (Marhamatizadeh
et al., 2012 d).
The results of the experiments in this work represent that Denak (Oliveria decumbens vent.) has suitable support for these intestinal bacteria for production of probiotic products that were kept viable up to the end of fermentation (21 days). All tested strains were proved a good growth capacity in Denak milk and yoghurt without nutrients added, this being a guarantee on the one hand for the normal evolution of the fermentation and on the other hand for the stability of the final product. Thus it seems that the nutrients are available in acceptable forms and in optimal concentrations in the tested Denak milk and yoghurt. Further, the viability of the probiotics is essential for the quality of the fermented dairy products. The survival of probiotic Denak milk and yoghurt in refrigerated conditions for at least 21 days was in number of greater than 109 cfu mL-1 which is essential if a product should have probiotic properties. It is important to emphasize that all the products possessed excellent stability during 21 days of storage. The sensory scores of the products were acceptance. From the foregoing results it can be concluded that Denak can be successfully used in formulation of dairy products.