Eur Food Res Technol DOI 10.1007/s00217-012-1798-4
ORIGINAL PAPER
Effects of Chlorella vulgaris and Arthrospira platensis addition on viability of probiotic bacteria in yogurt and its biochemical properties Hannane Beheshtipour • Amir Mohammad Mortazavian Parivash Haratian • Kianoosh Khosravi Darani
•
Received: 5 May 2012 / Revised: 7 July 2012 / Accepted: 23 July 2012 Ó Springer-Verlag 2012
Abstract It is a practice to add microalgae into plain and probiotic fermented milks in order to promote the functionality of these products via their direct health effects as well as the enhancing impact on viability of probiotic microorganisms in product and in gastrointestinal tract. In this study, the effects of addition of two species of microalgae including Arthrospira platensis and Chlorella vulgaris (seven yogurt treatments containing three concentrations for each microalgae—0.25, 0.50, and 1.00 %— and a control without microalgae) on pH, titrable acidity, and redox potential changes as well as on the viability of probiotic bacteria during fermentation and during a 28-day refrigerated storage period (5 °C) were investigated in yogurt. Also, the amounts of lactic and acetic acids at the end of fermentation were assessed. The culture composition of yogurt was ABY type, containing Lactobacillus acidophilus LA-5, Bifidobacterium lactis BB-12, Lactobacillus delbrueckii ssp. bulgaricus, and Stresptococcus themophilus. The addition of microalgae significantly (p \ 0.05) increased the viability of L. acidophilus and bifdobacteria at the end of fermentation and during the storage period. Treatments containing A. platensis had H. Beheshtipour A. M. Mortazavian (&) P. Haratian Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences, Food Science and Technology, Shahid Beheshti University of Medical Sciences, P.O. Box 19395-4741, Tehran, Iran e-mail:
[email protected];
[email protected] K. K. Darani Department of Food Technology Research, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences, Food Science and Technology, Shahid Beheshti University of Medical Sciences, P. O. Box 19395-4741, Tehran, Iran
slower pH decline, faster acidity increase, longer incubation time, and greater final titrable acidity than those containing C. vulgaris and control. In treatments containing 0.5 or 1 % microalgae, the viability was almost higher than 107 cfu/mL until the end of refrigerated storage. Keywords Chlorella vulgaris Probiotic A. platensis Yogurt
Introduction Nowadays, manufacture of fermented milks containing probiotic microorganisms is a common and popular issue with a commercial significance, and many products of this kind are available in markets of different countries [1–6]. Presently, the genera Lactobacillus and Bifidobacterium are frequently used in production of probiotic fermented milks. Among bifidobacteria, the species Bifidobacterium lactis (B. animalis ssp. lactis) is preferred to be used by manufacturers because of its good tolerance to detrimental environmental factors of fermented milks such as acid, low pH, and molecular oxygen [1, 7–9]. Viability of probiotic microorganisms in the final product until the time of consumption is their most important qualitative parameter. Although there is no worldwide agreement on the minimum of viable probiotic cells per gram or milliliter of probiotic product until the time of consumption, generally, the values of 106 and 107–108cfu/ mL or cfu/g have been accepted as the minimum and satisfactory levels, respectively. In Japan, the ‘Fermented Milks and Lactic Acid Bacteria Association’ have developed a standard that requires a minimum of 107 cfu/mL viable probiotic cells to be present in dairy products [1, 6, 10, 11]. In Iran, National standard requires minimum
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of 106 cfu/mL viable probiotic cells in yogurt [12]. It has also stated that probiotic products should be consumed regularly with an approximate amount of 100 g day-1 in order to deliver about 109 cfu/mL viable cells into the intestine [1]. Probiotic bacteria normally lose their viability during fermentation and storage period especially in fermented milks [2, 3, 7, 8, 10, 13–16]. Microalgae (Cyanobacteria or blue-green algae) are photosynthetic (photoautotrophic) microorganisms that can be used to produce high-value compounds. Spray-dried microalgal biomasses typically contain 3–7 % moisture, 46–63 % protein, 8–17 % carbohydrates, 4–22 % lipids, 2–4 % nucleic acid, 7–10 % ash, and a wide range of vitamins and other biologically active substances such as bioactive peptides and pigments [17–19]. Microalgae have been commercially produced, and the mainly used genera are Chlorella and Arthrospira for health food [17]. A. platensis is the best known genus because of its high protein content and its excellent nutritional value. It has been claimed that A. platensis has various possible healthpromoting effects: the alleviation of hyperlipidemis, suppression of hypertension, protection against renal failure, growth promotion of intestinal Lactobacillus, and suppression of elevated serum glucose level due to its chemical composition including compounds like essential amino acids, vitamins, natural pigments, and essential fatty acids, particularly c-linolenic acid, a precursor of the body’s prostaglandins [20–22]. Besides, it has also been reported to have antimicrobial activities against some pathogenic bacteria [23] as well as to promote the growth of lactic acid bacteria in synthetic media [24]. Moreover, similar effects of A. platensis have been also detected in milk and fermented milk [19, 20, 25]. Chlorella vulgaris is a green algal species that produces colorants including astaxanthin, canthaxanthin and, in minor amounts, b-carotene and lutein [27]. Although the technological impacts of A. platensis addition on growth of lactic acid bacteria and probiotic bacteria in milk and fermented milks have been investigated [18–20, 24–28], there is no research with a comparative approach on the effects of A. platensis and C. vulgaris addition in yogurt containing ABY-type culture composition. Therefore, in present study, the effects of A. platensis and C. vulgaris addition on microbiological and biochemical aspects of ABY probiotic yogurt were investigated.
Materials and methods Starter culture Fifty-unit pouches of commercial lyophilized ABY culture (containing Lactobacillus acidophilus LA-5, Bifidobacterium
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lactis BB-12, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus) that are known as ‘FD-DVS ABY-1’ were supplied by Chr-Hansen (Horsholm, Denmark). This culture that consists of 12 g lyophilized starter powder is currently used by dairy industry to produce probiotic dairy-fermented products. The cultures were maintained according to the manufacturer’s instructions at -18 °C until used. For each sample preparation, one pouch of starter culture was gently dissolved in one liter of sterilized skimmed milk, and a desired inoculum was inoculated into the definite volume of milk base for fermentation, in conformity with the manufacturer’s instructions for direct use in the industry. Study design and sample preparation Seven yogurt treatments containing different types of microalgae powders, A. platensis or C. vulgaris (FardaSabz Iranian Co., Tehran, Iran), in different concentrations (0.25, 0.50, and 1.00 %) were produced using reconstituted skim milk powder and sterilized potable water. Zero % concentration of each microalgae was recognized as control. Reconstituted milk samples were heat treated at 85 °C–30 min, and the microalgae powders were added after cooling of milk up to fermentation temperature. Fermentation was then carried out at 40 °C until pH reached 4.5 ± 0.02. Biochemical parameters including pH drop, acidity increase, and redox potential increase were monitored throughout the fermentation period. These parameters were recorded every 30 min. Other biochemical parameters (‘Chemical analysis’) were determined at the end of fermentation. The final samples were cooled down and kept at 5 °C for 28 days. Viability of probiotic organisms was determined at the end of fermentation and within the storage period per 7-day intervals. The amounts of lactic and acetic acids were assessed at the end of fermentation as well as at the end of storage period. The design of present study is shown in Fig. 1. Microbiological analysis MRS-bile agar medium (MRS agar by Merck, Darmstadt, Germany and bile by Sigma-Aldrich, Inc., Reyde, USA) was used for the selective enumeration of L. acidophilus and bifidobacteria in ABY culture composition according to the previous papers [8, 29]. The plates were incubated aerobically (for L. acidophilus) and anaerobically (for total probiotic bacteria) at 37 °C for 72 h. Anaerobic conditions were produced using the GasPac system (Merck, Darmstadt, Germany). Viability proportion index (VPI) of probiotic microorganism at the end of fermentation was calculated as following [2, 3, 16].
Eur Food Res Technol Reconstituted skim milk (12% MSNF)
Heat treatment (85°C-15 min)
Adding A. platensis
0.25%
0.50%
Adding C. vulgaris
1.00%
0.25%
0.50%
Control (free of icroalgae)
1.00%
Cooling down to fermentation temperature
Inoculation with ABY
Incubation at 40°C up to pH4.5
Experimental parameters during fermentation
Changes in pH, titrable acidity and redox potential, incubation time
Cooling up to 5°C
Experimental parameters at the end of fermentation
Parameters related to pH, titrable acidity and redox potential,
and during refrigerated storage period (28 d, 5˚C;7-day intervals)
Viability of probiotics, concentrations of lactic and acetic acids
Fig. 1 Study design of present study for single replication
VPI ¼ Final cell population ðcfu=mLÞ= initial cell population ðcfu=mLÞ Viability decrease/increase percentage of probiotic microorganism during the storage period was calculated as following [2, 3, 16]. Viability decrease=increase percentage ¼ Final cell population ðcfu=mLÞ initial cell population ðcfu=mLÞ= initial cell population 100 Chemical analysis pH values and redox potential of the samples were measured at room temperature using a potentiometer (MA235, Mettler, Toledo, Switzerland). The titrable acidity was determined after mixing 10 mL of sample with 10 mL of distilled water and titrating with 0.1 N NaOH using 0.5 % phenolphthalein [3, 10]. Various biochemical parameters were defined and determined as follows: Mean pH drop rate (mpH-DR) = (final pH number initial pH number)/incubation time [pH number/min] [2, 16] Mean acidity increase rate (mA-IR) = (final acidity value - initial acidity value)/incubation time [Dornic degree/min] [2, 16]
Mean redox potential increase rate (mRP-IR) = (final value - initial value)/incubation time [mV/min] [2, 16] Time to pH 5.5 (t5.5): time from the start of incubation until reaching the pH of 5.5 [min] Time of maximum pH drop (tmax-pH-D): The 30-min time interval during fermentation in which the greatest pH decline is observed [min–min] Time of maximum titrable acidity increase (tmax-A-I): The 30-min time interval during fermentation in which the greatest titrable acidity increase is observed [min– min] pH of maximum pH drop (pHmax-pH-D): The pH range within a 30-min time interval during fermentation in which the greatest pH decline is observed [pH value-pH value] Quantification of lactic and acetic acids was carried out by high-performance liquid chromatography (CE 4200Instrument, Cecil, Milton Technical Center, Cambridge CB46AZ, UK) according to the previous papers [2, 16]. Briefly, for extraction of acids, 4.0 g of sample was diluted to 25 mL with 0.1 N H2SO4 homogenized and centrifuged at 5,000g for 10 min. The supernatant was filtered through Whatman #1 filter paper and through a 0.20-lm membrane filter and was immediately analyzed. A Jasco UV-980 detector and a Nucleosil 100-5C18 column (Macherey–Nagel, Duren, Germany) were used. The mobile phase was 0.009 N H2SO4 at a flow rate of 0.5 mL/min.
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Sensory analysis A panel of nine panelists was used. They were trained in ‘National Nutrition and Food Technology Research Institute-NNFTRI’ (Tehran, Iran). The treatments were analyzed and compared using a scoring method that was based on the Iranian national standard [30]. The sensory parameters were flavor, oral texture and mouthfeel, nonoral texture (pouring, stirring, and scoopability), and appearance (color, syneresis, and homogeneity with respect to the surface and texture). Each of these parameters was scored on a five-point scale: 0 = inconsumable; 1 = unacceptable; 2 = acceptable; 3 = satisfactory; and 4 = excellent. The score for each sensory parameter was multiplied by the relevant coefficient, namely 6 for flavor, 3.5 for oral texture and feel in the mouth, 2 for appearance, and 1 for nonoral texture.
Control
7
Each treatment was produced three times, and each experiment was performed in duplicate. Experiments were set up using a completely randomized design. Data were subjected to analysis of variance, and comparison of the means was done using two-way ANOVA test from Minitab software at significance level of 0.05.
Results and discussion Biochemical characteristics of yogurts Figure 2 shows changes in pH drop, acidity increase, and redox potential increase during fermentation period in different treatments. As shown in this Figure, for all treatments, five distinguished phases could be observed, namely lag and pre-log phases (initial part of the charts with relatively low slopes), log phase (with considering higher slope), and late log and stationary phases (with significantly decrease in chart slop compared to previous phase). This observation was in correspondence with those reported by other researchers [3, 7, 10, 16]. For all
200
100.8
100
5 50
30
4.5
150
101.7
33 4.48 31.5
60
90
120 150 180 210 240 270
4
0
0
30
60
90
pH
A
RP
pH
200
pH
5.5
94.5
100
5 50
27.9
200 C-1
6.43
150 135 111.6
5.5
100
5 50
36
4.5
4.48 28
4.48 30.6
4 0
30
60
90
120 150 180 210 240 270
0
4 0
30
60
90 120 150 180 210 240 270
time (min) pH
A
0
time (min) RP
Fig. 2 Changes in pH drop, acidity (A) increase and redox potential (RP) increase during fermentation period in different treatments. C-0.25, C-0.5, and C-1 represent the treatments containing 0.25, 0.50,
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RP
6
pH
136
A(°D)/ RP(mv)
6.5
6
A
7
C-0.25
150
4.5
0
time (min)
7 6.42
120 150 180 210 240 270
A(°D)/ RP(mv)
30
time (min)
6.5
100
50
4.5
24.3
0
136
5.5 5
4.48
4
200
6.45
6
pH
5.5
6.5
A(°D)/ RP(mv)
150 136
6
C-0.5
7
6.36
6.5
pH
Statistical analysis
A(°D)/ RP(mv)
The wavelength of detection was optimized at 210 nm. The standard solutions of lactic and acetic acids (Merck, Darmstadt, Germany) were prepared in distilled water. The retention times for lactic and acetic acids were 3.45 and 3.58 min, and the standard curve regression coefficients were 0.989 and 0.991, respectively.
pH
A
RP
and 1.00 % C. vulgaris, respectively. S-0.25, S-0.5, and S-1 represent the treatments containing 0.25, 0.50, and 1.00 % A. platensis, respectively
Eur Food Res Technol 7 6.5
200
S-0.25 6.29
A°(D)/ RP(mv)
138 150
pH
6 120.6
5.5
100
5 36
50
4.5
4.5 30.6
4 0
30
60
90
0
120 150 180 210 240 270
time (min) RP
S-0.5
7 6.5
A
200
6.3
136
150
pH
6 122.4
5.5 5
36
100
50
4.5
A(°D)/ RP(mv)
pH
4.5 28.8
4
0
30
60
90
120 150 180 210 240 270
0
time (min) pH
RP
S-1
7
200
6.46 138
pH
6
150
124.2
5.5
100
5 4.5
4.5
27
50
A°(D)/ RP(mv)
6.5
A
29.7
4 0
30
60
90
120 150 180 210 240 270
0
time (min) pH
A
RP
Fig. 2 continued
treatments, the minimum decrease rate of pH as well as the minimum increase rates of acidity and redox potential was observed within the initial steps of fermentation that could be attributed to the being in the late lag/early log phases of bacterial growth as well as the relatively high buffering capacity of milk [3, 16]. As is evident in Table 1, the significantly (p \ 0.05) slower mean pH drop rates were observed for the treatments constituting A. platensis. These treatments also showed significantly greater mean acidity increase rates (p \ 0.05), except for C. vulgaris -0.5 %. In contrast, control showed significantly lower mean acidity increase
rates. Similar situations were observed for final acidity in treatments. These characteristics can be attributed to the different buffering capacity of the treatments. Samples containing A. platensis exhibited higher buffering capacity [31] probably due to the considerable enrichment of milk by proteins, peptides, and amino acids via addition of this microalga. Greater buffering capacity leads to slower pH drop and stimulates acidification rate by starter bacteria because they are inhibited considerably later during fermentation. Sharp decline in pH causes pH drop shock to the starter bacteria, especially probiotic bacteria [3]. The greatest mean acidity increase rate as well as the final titrable acidity among the treatments was observed for those containing A. platensis. As mentioned, this is related to the higher buffering capacity of media containing A. platensis. Time of maximum pH drop (tmax-pH-D) in treatments containing A. platensis was 120–150 (min–min), while this time for the control and treatments containing C. vulgaris was 150–180 (min–min) (Table 1). Similar specifications were observed for the time of maximum titrable acidity increase (tmax-A-I), indicating that during fermentation, starter bacteria in the treatments containing 1 % A. platensis are 30 min sooner than in the peak of acidification and activity. The data related to the pH of maximum pH drop (pHmax-pH-D) are in correspondence with the data related to the time of maximum pH drop in treatments. For example, treatment with 1 % of A. platensis showed 0.53 decline in pH (6.07–5.54) within the hours 120–150 during fermentation, while this pH decline value was 0.45 (5.62–5.17) in control (Table 1). This phenomenon indicates that in the treatment with 1 % of A. platensis, the greatest pH decline during fermentation started from higher pH value (6.07 compared with 5.62). According to Table 1, the time to pH 5.5 (t5.5) was not considerably different among treatments (not more than 7 min). Comparing the data related to this parameter with those related to pHmax-pH-D and tmax-pH-D reveals that the sharpest pH decline in treatments with A. platensis within hours 120–150 of fermentation compensates with the sharpest pH decline in treatments containing C. vulgaris within hours 150–180. On the other words, in treatments with A. platensis, the fastest pH-decline period (120–180 during fermentation) finished before pH 5.50, while in those with C. vulgaris, this period (150–180) includes pH 5.50. Considering Table 1, the longest incubation time was observed for the treatments containing A. platensis (270 min) in comparison with those containing C. vulgaris (240 min). Incubation time in control treatment was 245 min. Therefore, adding A. platensis powders to yogurt milk led to increase in incubation time compared to the control and the treatments containing C. vulgaris. Therefore, addition of A. platensis into milk led to considerably higher acidification rate and as a result,
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significantly shorter incubation time [17, 31]. In these treatments, considerable increase in buffering capacity of milk led to reduction of mean pH drop rate and longer incubation time than control. This observation corresponds with the data related to mean pH drop rate (Table 1) in which the treatments containing A. platensis had greater mean pH drop rate (0.008) compared to those containing C. vulgaris or control (0.007). With respect to Table 1, the greatest and lowest amounts of lactic acid were observed in treatments containing A. platensis (0.5 and 1.0 %) and control, respectively. There was no significant difference in acetic acid concentration in different treatments. According to Table 2, mean acidity increase rate, final acidity (d28), and final redox potential were significantly greater in treatments containing A. platensis than the others, indicating higher activity of starter cultures in mentioned treatments during refrigerated storage. However, no
significant difference for mean pH drop rate was found among the treatments. Viability of probiotic bacteria at the end of fermentation and during refrigerated storage Table 3 shows viability of probiotic microorganisms as well as the relevant viability proportion indexes (VPI) in different treatments immediately after fermentation. As represented from this table, the viability of both probiotic bacteria (L. acidophilus LA-5 and B. lactis BB-12) was significantly and markedly greater in the treatments containing microalgae than control. Also, the higher concentration of microalgae (from 0.25 to 1.0 %) had the greater viability of both probiotic bacteria at the end of fermentation. The greatest viability for both probiotic bacteria was observed in the treatments containing 1 % of A. platensis or C. vulgaris microalgae. According to Table 3, the
Table 1 Biochemical characteristics in treatments throughout the fermentation or at the end of this period Parameters Algae type
Percent
mpH-DR** (pH/min)
mA-IR (° D/min)
mRP-IR (mV/min)
t5.5 (min)
tmax-pH-D (min–min)
tmax-A-D (min–min)
pHmax-pH-D (pH–pH)
Incubation time (min)
Final acidity (°D)
Lactic acid (%)
Acetic acid (%)
Control
0
0.008a
0.31d
0.43c
158
150–180
150–180
5.62–5.17
245b
100.8d
0.94e
0.13a
a
e
a
b
e
d
C. vulgaris
0.25
0.008
0.28
0.46
156
150–180
150–180
5.61–5.08
240
101.5
1.17
0.12a
C. vulgaris
0.50
0.008a
0.30de
0.45ab
154
150–180
150–180
5.51–5.02
240b
103.7f
1.15d
0.12a
1.00
a
5.53–5.06
b
c
C. vulgaris A. platensis A. platensis A. platensis
0.008
b
0.25
0.007
b
0.50
0.007
b
1.00
0.007
c
cd
0.34
0.42
a
d
0.37
0.40
ab
de
0.36
0.39
a
c
0.37
0.43
152 159 152 153
150–180 120–150 120–150 120–150
150–180 120–150 120–150 120–150
5.96–5.60 5.95–5.53 6.07–5.54
240
a
270
a
270
a
270
111.6
a
124.2
ab
122.4
a
125.1
ab
0.12a
c
0.14a
a
0.13a
a
0.14a
1.39 1.35 1.43 1.42
Means in the same column shown with different letters are significantly different (p \ 0.05) ** mpH-DR = mean pH drop rate, mA-IR = mean acidity increase rate, mRP-IR = mean redox potential increase rate, t5.5 = time to pH 5.5, tmax-pH-D = time of maximum pH drop, tmax-A-I = time of maximum titrable acidity increase, pHmax-pH-D = pH of maximum pH drop
Table 2 Biochemical parameters in treatments during storage period or at the end of this period (28 days, 5 °C) Treatment
Parameters
Type
Percent
mpH-DR** (pH/day)
mA-IR*** (°D/day)
mRP-IR**** (mV/day)
Final acidity (day 28) (°D)
Final RP (day 28) (mV)
Control
0
0.001a
0.02f
0.03d
110.7de
137d
a
e
f
C. vulgaris
0.25
0.001
0.04
0.00
103.5
132e
C. vulgaris
0.50
0.001a
0.04e
0.02de
117.0d
139d
1.00
a
d
c
146c
b
152b
a
152b
a
164a
C. vulgaris A. platensis A. platensis A. platensis
0.25 0.50 1.00
0.001
a
0.001
a
0.001
a
0.001
0.10
c
0.17
b
0.19
a
0.21
c
0.05
c
0.05
b
0.06
a
0.10
e
135.0 169.2 180.0 180.0
Means in the same column shown with different letters are significantly different (p \ 0.05) ** mpH-DR = pH drop rate, *** mA-IR = acidity increase rate, **** mRP-IR = redox potential increase rate
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similar concentrations. Increasing the viability of B. lactis BB-12 was not significantly different in each similar concentration of A. platensis and C. vulgaris, except for 0.5 % of A. platensis, in which the viability of B. lactis was greater (p \ 0.05). According to Table 5, the ‘viability decrease/increase percentage’ for control decreased significantly at day 14 compared to day 7. However, in other treatments, the rate of this reduction was very slower. At day 21, adding 0.5 % of A. platensis increased the viability of both probiotic bacteria significantly more efficient than C. vulgaris. Incorporating 1 % of C. vulgaris increased the viability of L. acidophilus LA-5 (but not B. lactis BB-12) significantly more than A. platensis. However, adding 0.25 % of C. vulgaris and A. platensis during storage period showed almost similar changes. At day 21, the rate of viability decrease percentage was higher in control compared to microalgae-containing samples. At day 28, in all treatments with the same microalgae concentrations, A. platensis possessed significantly greater viability of both probiotics compared to C. vulgaris. Even though the greatest viabilities of both probiotic microorganisms obtained in treatments containing 1 % of microalgae, the concentration of 0.5 % was also sufficient to maintain the viability of probiotic microorganisms greater than 107 cfu/mL in most treatments until the day 21 of storage period. The treatment containing 0.25 % of C. vulgaris has the smallest viability. Our findings agreed with Molnar’s study [31], who determined that during the first 2 weeks of refrigerated storage at 4 °C, the Lactococcus counts were significantly higher (p \ 0.05) in the cyanobacterial fermented milk than in the control product, confirming the stimulatory effects of A. platensis on laboratory [31]. Varga reported during refrigerated storage, the A. platensis–supplemented-fermented ABT milk contained significantly higher (p \ 0.05) levels of lactobacilli than did the control product [19].
growth proportion index (VPI) for the treatments containing microalgae is significantly greater than control, especially for L. acidophilus LA-5 (81.87 and 79.37). The positive effects of microalgae on viability of probiotics can be attributed to the reason that microalgae provide higher nutritious and stimulatory media for lactic acid bacteria and probiotic bacteria and stimulate their growth and activity. From these substances, exopolysaccharide, adenine, hypoxanthine, free amino acids, and essential vitamins and minerals can be mentioned [24, 27, 28, 32–36]. Addition of A. platensis (compared with C. vulgaris) led to higher buffering capacity and longer fermentation time until reaching the final pH of fermentation (Table 1, section ‘Biochemical characteristics of yogurts’). Generally, lower buffering capacity of treatments results in sharper decrease in pH during fermentation and storage periods (greater mean pH drop rate), which causes pH drop shock to probiotic cells and shorter time of cell multiplication until the final pH of fermentation following by decrease in their viability [1, 2, 16]. In this study, although A. platensis-containing treatments had higher buffering capacity than those containing C. vulgaris, no significant viable counts were observed among them in most cases, indicating that providing a highly nutritious medium by adding C. vulgaris compensated the adverse effects of its lower buffering capacity (compared with A. platensis). Table 4 shows the viability of probiotic microorganisms in different treatments during refrigerated storage. The viability of both probiotic bacteria (L. acidophilus LA-5 and B. lactis BB-12) was significantly greater in the treatments containing microalgae compared to control, after one week of refrigerated storage (day 7). At this time, there was no significant difference in viability of probiotics between the two microalgae (at the same concentrations). At day 14, viability of L. acidophilus LA-5 in treatments containing 0.25 and 0.5 % of A. platensis was significantly greater than the treatments containing C. vulgaris with
Table 3 Viability of probiotic microorganisms and the viability proportion index in different treatments at the end of fermentation Treatment
Initial population (log cfu/mL)
Final population (day 0) (log cfu/mL)
VPI**
A?B
A
B
A?B
Type
Percent
A***
B****
A ? B*****
A
B
Control
0
6.21
6.44
6.64
7.36f
7.46f
7.71e
14.37
10.74
12.09
e
e
d
C. vulgaris
0.25
6.21
6.44
6.64
7.57
7.61
23.75
15.18
18.37
C. vulgaris
0.50
6.21
6.44
6.64
7.83bc
7.87c
8.15b
43.12
27.77
33.48
C. vulgaris
1.00
6.21
6.44
6.64
8.11a
8.19a
8.45a
81.87
58.14
66.97
A. platensis
0.25
6.21
6.44
6.64
7.69d
7.72d
8.02c
31.25
19.62
24.18
A. platensis
0.50
6.21
6.44
6.64
7.90b
7.92b
8.13b
50.62
31.11
32.09
6.64
a
a
79.37
51.48
61.86
A. platensis
1.00
6.21
6.44
8.10
7.89
a
8.143
8.42
Means shown with different English letters represent significant differences (p \ 0.05) in the same columns (among the treatments) ** VPI = Viability proportion index *** A = L. acidophilus, **** B = bifidobacteria, ***** A ? B = total probiotics
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Eur Food Res Technol Table 4 Viability of probiotic microorganisms in different treatments during refrigerated storage Treatment
Final population (day 7) (log cfu/mL)
Final population (day 14) (log cfu/mL)
Final population (day 21) (log cfu/mL)
Final population (day 28) (log cfu/mL)
A?B
A
A?B
A
Type
Percent
A**
Control
0
7.27e
C. vulgaris
0.25
7.59
cd b
B***
A ? B****
A
7.27f
7.60e
6.77g
6.90f
7.14g
6.47f
6.60e
6.84f
6.30a
6.30f
6.69g
e
d
f
e
f
e
d
e
a
e
6.97f
c
7.30d 7.57b
7.61
cd
7.90
B
7.41
7.47
7.74
B
7.27
7.35
c
d
d
g
d
c
7.59
d
6.68
B
d
A?B
6.66
C. vulgaris C. vulgaris
0.50 1.00
7.84 8.09a
7.85 8.16a
8.15 8.43a
7.65 8.00a
7.64 8.09a
7.94 8.35a
7.47 7.78a
7.46 7.80a
7.77 8.09a
6.99 7.25ab
7.01 7.30b
A. platensis
0.25
7.65c
7.66e
7.95d
7.47e
7.54e
7.81e
7.20e
7.36d
7.59e
6.80b
6.93
A. platensis
0.50
7.81b
7.90c
8.16c
7.77c
7.74c
8.05c
7.60bc
7.56b
7.88c
7.12c
7.25b
1.00
a
b
b
a
a
A. platensis
8.06
ab
8.07
8.35
7.92
ab
7.94
b
8.24
b
7.69
a
7.76
ab
8.03
7.38
cd
7.41
7.11e 7.49bc 7.69a
Means shown with different English letters represent significant differences (p \ 0.05) in the same columns (among the treatments) ** A = L. acidophilus, *** B = bifidobacteria, **** A ? B = total probiotics Table 5 Loss/increase percentage in viability of probiotic bacteria during 28 days of storage at 5 °C, per 7-day intervals (compared to the initial viable cell counts immediately after fermentation or the viable cell counts at the last days of each 7-day storage interval) Treatment
Storage time (day)
Type
Percent
Control
0
C. vulgaris C. vulgaris C. vulgaris
0.25 0.50 1.00
Probiotic
0–7
7–14
LP0/IP0***
LP0
14–21 LP7
LP0
21–28 LP14
LP0
LP21
A*
-18
-74
-69
-87
-50
-92
-33
B**
-24
-74
-65
-87
-50
-91
-29
A
?2
-32
-34
-50
-27
-88
-75
B
?1
-31
-32
-51
-21
-89
-76
A
?1
-39
-38
-57
-32
-86
-68
B
-2
-39
-38
-60
-33
-87
-67
A
-5
-23
-20
-54
-40
-87
-71
B
-7
-22
-17
-57
-45
-87
-70
-47
-88
-60 -67
A. platensis
0.25
A
-10
-40
-33
-68
B
-13
-37
-28
-63
-41
-88
A. platensis
0.50
A
-1
-28
-10
-51
-33
-84
-66
B
-2
-18
-22
-45
-33
-78
-60
A
?1
-34
-27
-61
-42
-82
-52
B
?2
-35
-25
-60
-38
-82
-54
A. platensis
1.00
* A = L. acidophilus, ** B = bifidobacteria *** LP0/IP0 = loss or increase percentages; Small numbers (0, 7, 14, 21, 28) represent reference points for cell count comparisons
Sensory characteristics of treatments at the end of fermentation Table 6 shows the sensory analysis of treatments using score methodology. As represents, treatments with higher amounts of microalgae possessed weaker sensory acceptability for all sensory parameters compared to the control. A. platensis exhibited more unpleasant flavor compared with C. vulgaris, and the treatment with 1 % of A. platensis had the lowest flavor score. The inappropriate flavor caused by addition of microalgae is related to the generated compounds from the oxidation of polyunsaturated fatty acids occurred in considerable amounts in their powder as
123
well as different minerals that not only act as pro-oxidants but also might produce metallic off-tastes [19–21]. Addition of microalgae into the yogurt changed the color of this product to greenish or bluish based on the type and concentration of microalgae added. This characteristic was realized as an inappropriate sensory attribute (appearance) by the panelists. Moreover, mostly in treatments with 1 % of microalgae, graininess caused by insoluble microalgae particles was recognized. There were not considerable differences among the treatments from nonoral texture points of view. However, differences were remarkable from oral-texture standpoint. Treatments containing 1 % of microalgae had the lowest sensory score for oral texture
Eur Food Res Technol Table 6 Sensory analysis of the treatments using score methodology Treatments
Control
Parameters** Flavor
Oral texture and feel in the mouth
Appearance
Nonoral texture
Total score
18a
10.5a
8.0a
3.0a
39.5a
b
b
b
a
32.9b
C. vulgaris 0.25 %
16
C. vulgaris 0.50 %
16b
7.0b
7.0b
2.7a
32.7b
C. vulgaris 1.00 %
e
5.5
c
5.4
c
2.5
b
25.4d
7.0
b
6.5
b
2.8
a
31.3c
7.6
b
7.0
b
2.8
a
31.4c
5.0
c
5.0
c
2.5
b
18.5e
A. platensis 0.25 % A. platensis 0.50 % A. platensis 1.00 %
12
c
15
d
14
6
f
7.0
7.0
2.9
Means shown with small letters represent significant differences (p \ 0.05) in the same columns ** Every data point is the mean of nine replications (nine panelists)
and mouthfeel. Overall, treatments with C. vulgaris obtained higher sensory score compared to A. platensis. There was no significant difference between the treatments containing 0.25 or 0.5 % of both microalgae.
Conclusion Results of this study revealed that adding microalgae to yogurt milk significantly increased and sustained the viability of L. acidophilus and Bifidobacterium lactis at the end of fermentation as well as during refrigerated storage. Also, the treatments containing A. platensis had slower pH decline, faster acidity increase, longer incubation time, and greater final titrable acidity. Even though the greatest viabilities of both probiotic microorganisms obtained in treatments containing 1 % of microalgae, the concentration of 0.5 % was also sufficient to maintain the viability of probiotic microorganisms greater than 107cfu/mL throughout the storage period. Also, this 0.5 % concentration results in more appropriate sensory characteristic of final product while is more cost-effective (compared to 1 %). Investigating the effects of microalgae with different concentrations on other strains of probiotics in various dairy products is suggested. Also, sensory studies in them the masking effects of different types and concentrations of flavoring agents/flavors on the taint of microalgae-enriched yogurt are considered have its special priority.
2.
3.
4. 5.
6.
7.
8.
9.
10.
11. Acknowledgments This study, from a M.Sc. thesis of National Nutrition and Food Technology Research Institute (Shahid Beheshti University of Medical Sciences, Tehran, Iran), was financially and operationally supported by this institute. 12.
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