3.1. Coagulation time and Titratable acidity
Coagulation of milk appears as a result of the precipitation of milk protein (casein) in acidic conditions at a pH of around 4.6 (Lee and Lucey, 2010). In Table (1), the treatment T4 which prepared by 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS has the longest coagulation time (4 hr:10 min) with increasing percentage by 22.75% followed by treatment (T3) prepared by 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin (4 hrs: 5 min) increasing percentage by 21.25%, than treatment (T2) prepared by 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS ( 3hr :50 min) increasing percentage by 4.79%. The lowest coagulation time was recorded for treatment (T1) which was prepared by 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus (3 hr: 45 min) with an increasing percentage of 3.29% compared to control treatment (coagulation time of control was 3.29). The variation in the coagulation time may be attributed to the antibacterial activity of probiotic bacteria and prebiotics (inulin and FOS) on the starter culture of yogurt and subsequent acid production (Akabanda et al., 2014).
Table 1
changes in coagulation time among different groups of yoghurt
Yogurt samples
|
Coagulation time
|
( Hr : min)
|
Increase (%)
|
C
|
3:34
|
0.00
|
T1
|
3:45
|
3.29
|
T 2
|
3:50
|
4.79
|
T 3
|
4:05
|
21.25
|
T 4
|
4:10
|
22.75
|
C: 2% yogurt starter cultures 1:1 (control).
T1: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus.
T2: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS.
T3: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin.
T4: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS.
During refrigerated storage (4ºC), the titratable acidity of yogurt samples in all treatment groups increased gradually as the storage period progressed (Table 2). The addition of inulin and (FOS) insignificantly affected the titratable acidity of yogurt samples. On the other hand, the addition of inulin and FOS increased the shelf life to 39 days in group 4 which was prepared by 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS compared to control treatment (C) which spoiled at 21 day of refrigerated storage. Treatment T2 which prepared by 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS and T3: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin spoiled at 32 days of refrigerated storage. While treatment T1: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus was spoiled at 28 days of refrigerated storage. EOSQ (2005) stated that the titratable acidity of yogurt should not increase by 1.50%. Guven et al. (2005) found that yogurt's titratable acidity was not affected by the addition of different ratios of inulin, and it was increased during storage.
Table 2
Titratable acidity (% lactic acid) in yoghurt samples during storage
Storage
(Day)
|
Titratable acidity (Mean ± S. E*)
|
C
|
T1
|
T 2
|
T 3
|
T 4
|
Zero
|
0.84 ± 0.02A
|
0.80 ± 0.04A
|
0.79 ± 0.01B
|
0.75 ± 0.03B
|
0.72 ± 0.01C
|
7
|
0.95 ± 0.03A
|
0.97 ± 0.03A
|
0.95 ± 0.03A
|
0.83 ± 0.02B
|
0.79 ± 0.01C
|
14
|
1.23 ± 0.03A
|
1.10 ± 0.02B
|
1.06 ± 0.05BC
|
0.90 ± 0.01C
|
0.85 ± 0.00D
|
21
|
S
|
1.16 ± 0.03A
|
1.14 ± 0.02A
|
1.06 ± 0.03B
|
0.94 ± 0.02C
|
28
|
S
|
S
|
1.22 ± 0.03A
|
1.17 ± 0.03B
|
1.08 ± 0.02C
|
32
|
S
|
S
|
1.34 ± 0.03A
|
1.26 ± 0.05B
|
1.18 ± 0.3C
|
39
|
S
|
S
|
S
|
S
|
1.20 ± 0.5A
|
C: 2% yoghurt starter cultures 1:1 (control). |
T1: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus. |
T2: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS. |
T3: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin. |
T4: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS. |
S: spoilage |
*The values indicated are the mean ± S.E (n = 3). Values in the same raw denoted by different letters (ABCD) differ significantly (p < 0.05) from each other. |
3.2. Sensory Evaluation
Yogurt products are beginning increasingly popular throughout the world (Aryana and Olson 2017). In addition to their health benefits, the texture of the product plays an important role in the consumer's acceptance of the product (Vital et al., 2015). The addition of inulin and FOS improved the sensory properties of the resultant yogurt samples. The result revealed that treatment (T2), 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS and treatment (T4), 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS) were the most highest accepted groups regarding to sensory evaluation (Figs. 1, 2 and 3). Our results are in line with those of Srisu et al. (2013), who reported that the addition of prebiotic could improve the physical and sensory properties of the yogurt. On the other hand, Cruz et al. (2010) explored that the addition of inulin may cause changes in yogurt quality attributes due to interactions between the functional ingredient and food matrix components. Inulin may provide yogurt's creamy mouthfeel and sweet taste (Donkor et al., 2007). The use of inulin in yogurt production as carbohydrate fat substitutes can improve the perception of color (Brennan et al., 2008).
3.3. Microbiological Quality
Table (3) shows the growth of Lactobacillus delbrueckii sub spp bulgaricus throughout the storage period in different groups. There is a decline in the growth rate of Lactobacillus delbrueckii sub spp bulgaricus in all examined samples. Treatment that contain inulin including treatment T2: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS and treatment T4: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS have the highest count at the end of the storage period. The Lactobacillus delbrueckii sub spp bulgaricus in treatment (T2) count was 9.27 ± 0.12 CFU/g at 32 days of refrigerated storage, while treatment (T4), the count was 9.52 ± 0.04 CFU/g at 39 days of refrigerated storage.
Table 3
Survival of Lactobacillus delbrueckii sub spp bulgaricus (log 10 cfu/g) in different yogurt groups during storage
Storage (Day)
|
|
|
Treatments
|
|
|
C
|
T1
|
T2
|
T3
|
T4
|
Zero
|
12.75 ± 0.15A
|
12.75 ± 0.03A
|
12.65 ± 0.05B
|
12.73 ± 0.07A
|
12.71 ± 0.02A
|
7
|
10.58 ± 0.04D
|
11.35 ± 0.01C
|
12.33 ± 0.05B
|
12.15 ± 0.11B
|
12.47 ± 0.09A
|
14
|
9.00 ± 0.02D
|
10.40 ± 0.02D
|
11.86 ± 0.1C
|
11.53 ± 0.11B
|
12.31 ± 0.03A
|
21
|
S
|
9.22 ± 0.03D
|
11.13 ± 0.09B
|
10.75 ± 0.06C
|
12.14 ± 0.05A
|
28
|
S
|
S
|
10.35 ± 0.04B
|
10.52 ± 0.04B
|
11.24 ± 0.05A
|
32
|
S
|
S
|
9.27 ± 0.12C
|
10.12 ± 0.03B
|
10.53 ± 0.11A
|
39
|
S
|
S
|
S
|
S
|
9.52 ± 0.04A
|
C: 2% yogurt starter cultures 1:1 (control).
T1: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus.
T2: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS.
T3: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin.
T4: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS.
S: spoilage
*The values indicated are the mean ± S.E (n = 3). Values in the same raw denoted by different letters (ABCD) differ significantly (p < 0.05) from each other.
At the same time, the growth of Streptococcus thermophiles was also declined throughout the refrigerated storage. However, groups containing inulin and FOS have the highest count of Streptococcus thermophiles.
The count of Streptococcus thermophiles treatment (T2) was 7.65 ± 0.04 CFU/g at 32 days of refrigerated storage, and in treatment (T4), the count was 7.72 ± 0.04 CFU/g at 39 days of refrigerated storage (Table 4). These results agree with those obtained by EL-Nagar et al. (2002), and Akalin et al. (2007) mentioned that inulin could favor the growth and viability of lactic acid bacteria during fermentation or refrigerated storage. Furthermore, inulin concentrations were found to be perfect for stimulating growth and retaining the viability of probiotic cultures in fermented milk (Akalin et al., 2007; Aryana et al., 2007).
Table 4
Survival of Streptococcus thermophilus (log 10 cfu/g) in different yoghurt groups during storage
Storage (Day)
|
|
|
Treatments
|
|
|
C
|
T1
|
T2
|
T3
|
T4
|
Zero
|
9.77 ± 0.05A
|
9.54 ± 0.03A
|
9.76 ± 0.03A
|
9.62 ± 0.01A
|
9.73 ± 0.02A
|
7
|
8.50 ± 0.05C
|
9.11 ± 0.03AB
|
9.24 ± 0.01B
|
9.35 ± 0.02B
|
9.55 ± 0.01A
|
14
|
7.16 ± 0.06D
|
8.16 ± 0.03C
|
9.01 ± 0.01A
|
8.43 ± 0.01B
|
9.00 ± 0.01A
|
21
|
S
|
7.24 ± 0.05C
|
8.87 ± 0.03A
|
8.01 ± 0.03B
|
8.73 ± 0.03A
|
28
|
S
|
S
|
7.98 ± 0.03B
|
7.68 ± 0.04C
|
8.15 ± 0.04A
|
32
|
S
|
S
|
7.65 ± 0.04B
|
6.63 ± 0.04C
|
7.78 ± 0.04A
|
39
|
S
|
S
|
S
|
S
|
7.72 ± 0.04A
|
C: 2% yogurt starter cultures 1:1 (control).
T1: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus.
T2: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS.
T3: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin.
T4: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS.
S: spoilage
*The values indicated are the mean ± S.E (n = 3). Values in the same raw denoted by different letters (ABCD) differ significantly (p < 0.05) from each other.
Table 5 shows the mean Lb. acidophilus counts for the yogurt samples examined. Lb.acidophilus counts declined throughout the storage period in T1 (yogurt with Lb. acidophilus) and T2 (yogurt with Lb. acidophilus, 2% inulin, and 2%FOS) with the final counts of Lb. acidophilus was 7.54 ± 0.26 CFU/g at day 21 and 7.32 ± 0.26 CFU/g at day 32 of storage in T1 and T2, respectively. The addition of inulin effectively increased the Lb. acidophilus mean count when compared to the group that contains Lb. acidophilus without inulin. These results are in agreement with those obtained by Donkor et al. (2007c), Buriti et al. (2010), Hernandez-Hernandez et al. (2012) they found that prebiotic ingredients such as inulin and FOS may exert a protective effect and improve the survival and activity of probiotic bacteria during storage of probiotic food products as well as during the passage through the GIT.
Table 5
Survival of Lactobacillus acidophilus (log 10 cfu/g) in different yogurt groups during storage
Storage (Day)
|
Treatments
|
|
T1
|
T2
|
Zero
|
10.49 ± 0.11A
|
11.65 ± 0.08A
|
7
|
9.68 ± 0.06B
|
11.18 ± 0.06AB
|
14
|
8.43 ± 0.06C
|
9.43 ± 0.18B
|
21
|
7.54 ± 0.26D
|
8.84 ± 0.26C
|
28
|
S
|
7.52 ± 0.26D
|
32
|
S
|
7.32 ± 0.26D
|
39
|
S
|
S
|
C: 2% yoghurt starter cultures 1:1 (control).
T1: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus.
T2: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS.
T3: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin.
T4: 1% yoghurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS.
S: spoilage
*The values indicated are the mean ± S.E (n = 3). Values in the same column denoted by different letters (ABCD) differ significantly (p < 0.05) from each other.
The viability of probiotics was reported to be affected by many factors such as storage time, oxygen content, and fluctuation in temperature, low pH, reduced water activity, and high concentration of salutes (Carvalho et al., 2004 and Capela et al., 2006).
Our study observed that the addition of inulin to probiotic yogurt enhances the growth of S. thermophiles, L. bulgaricus, and L. acidophilus until the end of the storage period compared with groups without inulin. The increase in probiotic counts of yogurt may be attributed to the action of inulin as a prebiotic substance. Akın et al. (2007), Aryana et al. (2007), Sendra et al. (2008) attributed the increase in probiotic counts to the ability of probiotics and yogurt starter cultures to utilize inulin. Similarly, Donkor et al. (2007) showed that chicory-based inulin was a preferred carbon source for probiotic bacteria by increasing the growth performance and maintaining viability during cold storage. Acidity is one of the most critical factors that affect the viability of S. thermophiles, L. bulgaricus, and L. acidophilus (Dave and Shah, 1997). Oligosaccharides as (FOS) act as substrate for the growth of LAB and inhibition of colonic cancer cells growth and putrefactive or pathogenic bacteria present in the colon through the production of short chain fatty acids (SCFA). In addition, strengthening of the gut mucosal barrier, modification of gut microflora, prevention proliferation of pathogen and adherence to intestinal mucosa, and transformation of bacterial enzyme activity (Dma et al., 2009). Rousseau et al. (2005) reported that FOS could stimulate the growth of the beneficial strains but not pathogenic one. The inhibitory effect against pathogenic bacteria is usually as a result to reduction in pH results from acid production, secretion of hydrogen peroxide, and release of natural antibiotics (bacteriocin) from beneficial microflora selectively stimulated by various prebiotics (Manning et al. 2004).
In our study, (T4) yogurt samples appear to be the group that can resist spoilage with yeasts and molds (Table 6). According to EOSQ (2005) molds and yeasts, the count must not exceed 10 cfu/g in yogurt. T4 was in a permissible limit until 28 days of storage with a mean count was 1.00 ± 0.03 CFU/g, while T2 was within the permissible limit until 14 days of storage. T3 was within the permissible limit until 21 days of storage, with a mean count was 1.00 ± 0.05 CFU/g. T1 was within the permissible limit until 14 days of storage, with a mean count was 1.00 ± 0.01. At the same time, the control group, yeast, and mold were not detected in zero day only. These findings are consistent with the findings obtained by Abee et al. (1995), who mentioned that LAB produces a proteinaceous antimicrobial substance known as bacteriocins that generally act through inactivation of enzymes, depolarization of the target cell membrane, or inhibition of the formation of the cell wall of pathogenic microflora including bacteria, mold, and yeast. Probiotic bacteria also produce metabolic substances in varying quantities that may exert their antimicrobial effect by interfering with maintaining the cytoplasmic membrane, inhibiting active transport, and hindering various metabolic functions. Surajit et al. (2016) reported that inulin has antimicrobial activity against pathogenic microorganisms.
Magnusson and Schnürer (2001) and Rouse et al. (2008) mentioned that lactic acid bacteria produced antifungal metabolites that are proteinaceous. Lactobacillus acidophilus has the most significant activity against Aspergillus flavus and Aspergillus parasiticus. In addition, Lactobacillus acidophilus has been shown to reduce aflatoxin production during 30 days of storage at room temperature in maize kernels (Elsanhoty, 2008). The antifungal activity appears to be more assertive at lower pH ranges (Rouse et al., 2008); this may explain the antifungal activity of Lb. acidophilus bacteriocin in acidic yogurt media. Batish et al. (1990) and Stiles et al. (2002) reported that Lb. acidophilus has antifungal activity.
Coliforms counts were not detected in any of the yogurt groups samples on either the first day or during the storage period; this may be attributed to the good hygienic conditions during the preparation and storage of yogurts.
Table 6
Yeasts and moulds count (log 10 cfu/g) in different yogurt groups during storage
Treatments
|
|
|
|
Storage (Day)
|
|
|
Zero
|
7
|
14
|
21
|
28
|
32
|
39
|
C
|
ND
|
2.15 ± 0.01B
|
3.64 ± 0.04 A
|
S
|
S
|
S
|
S
|
T1
|
ND
|
ND
|
1.00 ± 0.01 B
|
2.36 ± 0.03 A
|
S
|
S
|
S
|
T2
|
ND
|
ND
|
ND
|
1.15 ± 0.02C
|
1.87 ± 0.01B
|
2.16 ± 0.03A
|
S
|
T3
|
ND
|
ND
|
ND
|
1.00 ± 0.05 C
|
1.35 ± 0.05 B
|
2.14 ± 0.06 A
|
S
|
T4
|
ND
|
ND
|
ND
|
ND
|
1.00 ± 0.03 C
|
1.85 ± 0.01B
|
2.01 ± 0.05 C
|
C: 2% yogurt starter cultures 1:1 (control).
T1: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus.
T2: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus + 2% inulin + 2% FOS.
T3: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin.
T4: 1% yogurt starter cultures 1:1 + 1% Lb. acidophilus bacteriocin + 2% inulin + 2% FOS.
S: spoilage
ND: Not Detected
*The values indicated are the mean ± S.E (n = 3). Values in the same raw denoted by different letters (ABC) differ significantly (p < 0.05) from each other.