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Improving the viability and stability of starter culture and the quality of fermented milk using some food additives.

Introduction

Fermented dairy products have been a major part of the diet of people around the world. Numerous scientific papers and review articles Hughes and Hoover (1991) and Molder, et al., (1990) have been published on the health benefits associated with the consumption of fermented dairy products. Over the past decade, considerable interest has developed in the use of probiotic organisms (Lactobacillus casei and Bifidobacterium spp.) in food, pharmaceutical, and feed products. Several therapetiuc benefits of probiotic bacteria have been claimed to be associated with the consummation of fermented milk product. The consumption of probiotic products has increased dramatically and >90 products containing L. acidophilus, or bifidobacteria, or both are available in the market, IDF (1984 and 1988). Suggested minimum numbers of probiotic bacteria at consumption are 105 to 106 cfu/g (Kurmann and Rasic 1991; Robinson, 1987).

Probiotic bacteria grow slowly in milk because of a lack of proteolytic activity Klaver, et al., (1993), and the usual practice is to add yoghurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus) to reduce the fermentation time.

Several factors affect the viability of probiotic bacteria (Klaver et al., 1990 Klaver, et al., 1993) Lankaputhra and Shah (1995) Lankaputhra, et al., (1996) and Proulx, et al., (1994). An increase in the acidity of the product during storage adversely affects the viability of probiotic bacteria. Hydrogen peroxide, produced by some lactobacilli, is known for its antimicrobial effects. Bifidobacteria are anaerobic in nature, and, therefore, higher oxygen content may affect their growth and viability. Antagonism among the bacteria used in the starter culture caused by the production of antimicrobial substances such as bacteriocins may decrease the numbers of sensitive organisms that may be present in a product or starter culture. In earlier studies, low viability of bifidobacteria was reported (Dave and Shah. 1997) in yoghurt made with one of the four commercial starter cultures. In those studies, a dramatic decline in the numbers of bifidobacteria in the same starter culture was observed, and higher concentrations of inoculums (Dave and Shah. 1997) or use of ascorbic acid as an oxygen scavenger did not celery improve the viability of bifidobacteria to a satisfactory level. No antagonism was observed between the yoghurt bacteria and the probiotic bacteria used in this starter culture (Joseph et al., 1998).Thus, milk base formulation and starter culture design seem to have strong effects on growth and stability of probiotic bacteria.

Several works have been done to improve the growth of probiotic bacteria. For instance, with pure culture of probiotic lactobacilli, Saxena et al., (1994) and Oliveira et al., (2002) noticed that acidification rate was doubled by adding casitone 5 g/kg -.1 or tryptone 20 g/L-.1. According to Shah and Ravula (2000), with a sugar level of 12% instead of 8%, the viability of probiotic cultures is lower (loss of 3 log instead of 2 log after 49 d of storage). For two strains of bifidobacteria, Shin et al., (2000) reported a better viability after 4 wk of storage when milk is supplemented with 5% of fructooligosaccharides. However, L. delbrueckii ssp. bulgaricus also produces lactic acid during refrigerated storage, known as post acidification, which is claimed to affect the viability of probiotic bacteria. To avoid the problem of postacidification, the present trend is to use starter cultures that are avoid of L. delbrueckii ssp. bulgaricus such as CBT (L.casei, bifidobacteria, and S. thermophilus). Such starter cultures may necessitate the incorporation of micronutrients (peptides and amino acids) through casein hydrolysate (CH), concentrate whey protein concentrate (WPC), and glucomacrpeptide (GMP), for reducing the fermentation time and for improving the viability of probiotic bacteria.

The objective of this study was to examine the effects of L-Ascorbic acid, CH, WPC and GMP, on the growth and viability of probiotic bacteria in fermented milk made with L.casei, Bifidobacteria and S. thermophilus (CBT) starter culture. Changes in pH, and viable counts of S. thermophilus, L. casei, and Bifidobacteria breve were monitored during manufacture and storage of fermented milk samples for 21 days at 5[degrees]C. Also, rheolgical, syneresis, biochemical and organolyptic properties were evaluated.

Materials and Methods

Materials: Starter Cultures

Streptococus thermophilus (S. thermophilus) ER1, Lactobacilus casei, (L.casei) (DSM 92157) and Bifidobaceria breve (B. breve) (DSM 20213) were obtained from BfEL, Kiel, Germany. The organisms were characterized, and identifies using specific PCR primers and by sugar fermentation patterns. After procurement, the starter cultures were stored at -20[degrees]C. They were thawed and diluted 10 times in sterilized milk or medium before use.

The dairy ingredients have been tested: concentrates whey protein concentrates WPC (Difco chemical Co.) and GMP was isolated by Dr.Chr. Lorenzen BfEL, Kiel Germany, casein hydrolysates CH (Difco chemical Co.). L-Ascorbic acid and skim milk powder were obtained from (Oxiod chemical Co.).

Fermented milk preparation

Fresh cows milk was from the herd of Faculty of Agriculture, Suez Canal University cooled to 4 [degrees]C and divided into 5 portions: the first portion was fortified with 1% skim milk powder and regarded as control, the second portion (T1) was fortified with 1 % skim milk powder and L-Ascorbic acid at level 200mg/kg, the third portion (T2) was fortified with CH at level 1% (wt/vol), the fourth portion (T3) was fortified with CWP at level 1% (wt/vol), the fifth portion (T4) was fortified with GMP at level 1 % (wt/vol).The treated milk was heated to 85[degrees]C for 15 min and then cooled to 42[degrees]C, followed by addition of CBT starter culture (2% of treated milk). The treated milk was distributed in 250-ml glass cups. Incubation was carried out at 37[degrees]C, and fermentation was terminated at pH 4.5. The time taken to reach pH 4.5 was recorded for each sample. After fermentation, samples were removed and stored at 5[degrees]C. Three replicates of each treatment were conducted.

The 0-h time started just after the addition of CBT starter cultures into the heattreated and cooled 42[degrees]C treated milk that were supplemented with various ingredients. The first day of storage carried out after overnight cold storage of stored fermented milk samples, analyses of fermented milk samples was cared out after 1, 7, 14, and 21 days of storage at 5[degrees]C, respectively.

Chemical Analysis

pH values of the fermented milk samples were measured by using a pH meter (Jenway pH meter electrode No. 29010, Jenway limited, England).. Protein content and total nitrogen was analyzed using Kjeldahal method as described by Ling (1963). Total solids and fat content were determined by standard methods (Marshall, 1992).

Viable Counts

A 1g sample was diluted with 9 ml of Ringer solution (Oxoid). Subsequent serial dilutions were prepared, and viable numbers were enumerated using the pour plate technique. Counts of S. thermophilus, L. casei, L.bulgaricus and B. breve were enumerated on LTM17 agar, MRS salicin agar, and MRS agar (Oxoid Chemical Co.) supplemented with lithium chloride, sodium propionate and paromomycin sulfate (Sigma Chemical Co.) for bifidobacteria. The colony-forming units (cfu) were converted to log10 and the results are reported as average of three replicates.

Syneresis

Synersis of fermented milk was measured using the drainage and centrifugation methods as described by Abou El-Nour et al., (2004). The volume of whey sepatred in different samples was measured and expressed in percent.

Rheological properties

Fermented milk was stirred for 5-min to achieve a visually homogenous slurry (Lankes et al., 1998). The rheological properties were measured using a Brookfield viscometer (Brookfield Engineering Laboratories Inc., MA, USA), equipped with a SC4-21 spindle running at 25 rpm. Measurements were made in the temperature of 25oC in shear rate ranging from 23.3 to 232.5 S-1.The measurements was repeated three times.

Biochemical analysis

Sugars in fermented milk was measured by HPLC analysis of all samples using a action exclusion column (OAKC-70, 7.5x300 mm, MERCK,) Pump A type:L-6200 at 70 [degrees]C. As mobile phase 0.0085 N H2SO4, at a flow rate of 0.4 ml/min, was used. Standard solutions of glucose, lactose and galactose, were included in the analysis. The sugars used for the standard curves were HPLC grade and were obtained from sigma Chemical Co. Samples preparation: one gram sample of fermented milk was blended with 3 g of 0.0085 N H2So4. The slurry was centrifuged at 5000 xg for 10 min at 4 [degrees]C. The supernatant was then filtered through a 0.45 [micro]m membrane filter (Sartorius, Minisart Co.) into vials and stored at 4[degrees]C into the Auto injector.

Organoleptic properties

Organoleptic assessment of fermented milk was evaluated by score panel of some of staff members of the dairy department faculty of Agriculture, Suez Canal University. Organoleptic assessments included flavour (10), color & appearance (5), body & texture (5) and over all acceptability (20). Organoleptic assessments were carried out according to Abou El-Nour et al., (2004).

* All results are average of three replicates.

Results and Discussion

Chemical Composition:

The total solids contents were in the range of 13.34 to 13.40 % for all products. The protein content was higher in fermented milk supplemented with WPC, CH and CMP respectively, and lowers in fermented milk supplemented with L.Ascorbic acid than the control. There were very little differences in the fat content of the all treatments of fermented milk.

Control: Fermented milk fortifited with 1% skim milk powder (control), T1: Fermented milk supplemented with 1 % skim milk powder and L.Ascorbic acid at level (200mg/kg-1), T2:Fermented milk supplemented with whey protein concentrates WPC 1 % (wt/vol),T3:Fermented milk supplemented with casein hydrolysates CH 1% (wt/vol), T4:Fermented milk supplemented with caseinomicropeptide GMP 1 % (wt/vol).

Changes of fermented milk during manufacture

I-Changes in pH

Changes in pH during the fermentation of fermented milk are presented in Fig (1).The decrease in pH was faster in fermented milk containing CH, WPC or GMP than that of the control one. Samples with added L.Ascorbic acid showed a drop in pH during fermentation that was quite close to that of the control fermented milk. However, increase in acidification rate was more pronounced for fermented milk fortified with GMP, CH, and WPC, respectively. Overall, the time taken to reach a pH 4.5 was 250 and 240 min for the control fermented milk and for the fermented milk supplemented with L.Ascorbic acid, respectively. On the other hand, the fermentation time decreased to 210 min for fermented milk supplemented with CH, and 220 min for that supplemented with WPC and 180 min for GMP, respectively. These data in agreement with data obtained by Champagne et al., (1996) concluded that WPC could be successfully used to prepare starter cultures because it gave higher populations of bacteria than when milk was used as a medium. Adding of WPC with milk protein hydrolysate also stimulated starter growth.

[FIGURE 1 OMITTED]

Control: Fermented milk fortifited with 1% skim milk powder (control), T1: Fermented milk supplemented with 1 % skim milk powder and L.Ascorbic acid at level (200mg/kg-1), T2:Fermented milk supplemented with whey protein concentrates WPC 1 % (wt/vol),T3:Fermented milk supplemented with casein hydrolysates CH 1% (wt/vol), T4:Fermented milk supplemented with caseinomicropeptide GMP 1 % (wt/vol).

Survival of starter culture bacteria during manufacture and storage of fermented milk: I-During manufacture

Changes in the counts of (CBT) starter culture during fermentation of fermented milk are presented in (Fig 2 a, b and c). Counts of S. thermophilus remained lower in contorol and fermented supplemented with L-Ascorbic acid, however CH, WPC and GMP supported the growth of S. thermophilus, and multiplication of this organism was faster in fermented milks supplemented with these ingredients, which could have been the reason for the shorter incubation time needed to reach pH of 4.5 for these samples.

When the time to reach pH 4.5 is taken into consideration and the counts of L. casei (Fig 2, b) are compared, the L. casei counts increased in the fermented milk that was supplemented with L.Ascorbic acid. Overall, multiplication of L. casei was faster in fermented milk that had been supplemented with CH, WPC and GMP.

Bifidbacteria throughout the fermentation process of the control slightly increased and then declined. During a 3-hrs period, their numbers slightly reduced followed by clear reduction at the end of manufacture. A similar trend of decreases was observed during the fermentation process for fermented milk supplemented with L.Ascorbic acid (T1). On the other hand, the bifidbacteria count increased and kept stable during manufacture when the fermented milk was supplemented with CH, WPC and GMP. Also, bifidobacteria counts were over <106 cfu/g throughout the incubation in fermented milk supplemented with CH, WPC and GMP (T2, T3 and T4). Lower redox potential was not solely responsible for improving the viability of bifidobacteria, but the additional nitrogen source in the form of peptides or amino acids was required to keep bifidobacteria viable in the product during manufacture of the fermented milk. The pH levels of all samples were kept almost identical during fermentation because low pH or higher acidity has been reported to affect the viability of probiotic bacteria (Kailasapathy and Supriadi 1996).

II-During Storage

There were no clear differences between the controls fermented milk and fermented milk sublimated with L-Ascorbic acid (T1) in the viable counts of S. thermophilus, L.casei and B.breve. While a clear increased in the viable counts of CBT start culture was observed in fermented milk supplemented with CH, WPC and GMP (T2, T3 and T4). The viable numbers of S. thermophilus was adversely affected, the counts in all treatments was slightly increased and slowly decreased during storage period. This decrease which more pronounced in all treatments after one week of storage may be due to the increase of the acidity percentage. On the other hand, the counts of L. casei were considerably higher and the viability in (T2, T3 and T4). Conversely, for other products, a shorter fermentation time might not have allowed L. casei to multiply to a greater extent, resulting. Thus, the pH of all fermented milk samples stabilized within this range, and the drop in pH did in lower counts of L. casei in finished products prepared with CH, WPC and GMP. Overall, the viability of L. casei was better in fermented milk supplemented with CH and GMP; however, counts remained >106cfu/g in (T2, T3 and T4) throughout the 21 days of refrigerated storage. Kailasapathy and Supriadi (1996) examined the effect of WPC on the survival of L. casei and concluded that the partial replacement of dried skim milk by WPC enabled sufficiently high numbers of L. casei to remain viable during 21 day of refrigerated storage.

The viability of bifidobacteria was low throughout the storage period for the control fermented milk and for the fermented milk supplemented with L.Ascorbic acid (T1).The viability of this organism in fermented milk supplemented with CH, WPC or GMP was improved by >2 log cycles compared with that of the control fermented milk. The highest viability of bifidobacteria was observed in fermented milk supplemented with CH and GMP (T2 and T4).

Improved viability could be due to the amino nitrogen present in CH, WPC, and GMP. The reduction in the total fermentation time from the addition of these ingredients and the favorable effects of micronutrients present in these ingredients might have been responsible for the improved viability of bifidobacteria to >106 cfu/g. This result showed that peptides and amino acids have improved the viability of probiotic bacteria, especially bifidobacteria, which have been reported to be weakly proteolytic.

Thus, addition of 0.5 % (w/vol) GMP may be sufficient and may be commercially feasible for improving the viability of probiotic bacteria. The addition of WPC and CH, however, may be more economical than the addition of GMP.

Changes in pH during storage:

Changes in pH during the refrigerated storage of fermented milk samples are shown in Fig (3). Approximately a 0.25-unit drop in pH was observed in the control treatment. The drop in pH in T2, T3 and T4 was highest than that observed in the control. T2 showed a similar trend of decrease in pH to that of control fermented milk. Changes in the pH of T2, T3 and T4 were similar and have the same trend. Overall, maxima of [??]0.27-unit drop in pH and were observed during 21 days of storage for T2, T3 and T4 possibly because of the availability of an amino nitrogen source through the added ingredients. Thus, the pH all fermented milk samples stabilized within this range, and the drop in pH did not seem to be factors that affected the viability of the bifidobacteria during refrigerated storage of fermented milk supplemented with these ingredients.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Curd syneresis

The syneresis values Table (2) measured by either the drainage or centrifugation mothods showed that fermented milk fortified with CH, WPC and GMP (T2, T3 and T4) respectively, exhibited less syneresis than that fortified with L-Ascorbic acid (T1) and control one. Fermnted milk fortified with WPC showed the lest syneresis,whereas the control batches the highest. Whey synersis values decresed in the following order WPC, GMP, CH, L-Ascorbic acid and the control respectively, using the drainge and centrifugation method which showed a smilar trend. This may be because WPC contain higer amount of denatured whey proteins as compared to GMP or CH which baounds more water resulting in low whey separation (Haret et al., 2003). Also the increase in the TS of fermented milk may explain also high firmness and low synersis of this product. The syneresisfor all treatments increased along the storage period as the development of acidity resultin mor agregation of casein micills.Syneresis depends on the water holding capacity of proteins, which depends on the protein content and the type of protein,the most important structural of fermentd milk is the strength of the coagulum and its ability to binding water (Abou-El-Nour et al., 2004).

Rheological properties

Table (3) showed the viscosity and flow index of fermented milk supplemented with various ingredients during cold storage.Addation of differnt ingredints in T2, T3 and T4 increased the fermrnted milk viscosity. Howver, the control sample and T1 showed lower viscosity. The viscosity was the highest in samples made by incorporating WPC, and the viscosity gradually decreased with GMP, CH, L-Ascorbic acid and control, resepectively. Viscosity was higer in products supplemented with WPC compared to other treatments because WPC has higher amount of protein, which may have contributed to the firmness of the product and resulted in the production of a more viscous gel. The rellations and trends sustained in the sample during the whole storage period.

The same table shows the flow index behaviour of fermented milk samples during storage at 5[degrees]C. Fermentd milk fortifitied with WPC (T3) was lower than fermented milk from other treatments.Manfactuer of fermented milk from milk fortifited with CH, WPC and GMP (T2, T3 and T4) reduce the flow behaviour of resultant samples than the control and T1 samples.

Carbohydrate analysis

Table (4) indicates that during storage period for 21 days of fermented milk fortifitited with viarus ingredints,the lactose concentartion decreased in all treatment, however T4 showed the highest level of decrese. Lactose concentartion decreased in the following order GMP<WPC<CH< L-Ascorbic acid and the control.Wherase, the rate of decreased in glucose concenrtion showed a smilar trend. The decrease in lactose concentration was associated with a decreased in glucose and increase in galactose. It seems that the cohice of fermented milk starter and type of additives is an important factor that determines starter cultuer activity during fermented milk manfactuer. Gekas and Lopez-Levia (1987) reported that the transglactosylase reaction varied with the enzym origin, the substrate copncentration, the type of substrate, the recactoin time, and other physico-chemical factors. Generally, during storage, lactose concentration for all treatmnts decreasesd resulting in an accumaltion of galactose, wherease the glucose concentartion decreased slightly.

Organoleptic properties

The average results of the organoleptic assesment of fermrnted milk fortifiteied with various ingredints in terms of flavour body and texture and appearnce are shown in table (5). Data showed that,at one day storage no clear differences was recorded in flavor for the treatments control, T1, T2 and T3. Fermented milk made from milk fortified with GMP T4 recorded the highest score for flavour during storage time than other treatments. However, prolonged storage period to 14 and 21 days decreased the flavour score. This was due to post acidification of fermented milk.

The effect of addition of various ingredients was more apparent on body and texture of fermented milk. Fermented milk supplemented with WPC and GMP recorded the highest score for body and texture, resultant fermented milk showed compact and soft body and texture than the other treatments. Control and T1 recorded the lowest score of body and texture. The color and appearance scores of all fermented milk were nearly the same after one day of storage. Control and T1 received the lowest score for color and appearance compared to other treatments. Fermented milk fortified with CH, WPC and GMP (T2, T3 and T4) resulted in fermented milk had a similar appearance properties. Resultant fermented milk of (T2, T3 and T4) gave fermented milk with high score for appearance, compared to other treatments as it revealed a dry, smooth and whiter shining surface. The total score revealed that fermented milk fortified with WPC and GMP gained the highest score for flavour, body &texture and color and appearance up to 21 days of storage at 5[degrees]C.

Generally, data showed that the fermented milk tasted mildly, comparable to well-fermented "yoghurt mild," and had a rich aroma. Nevertheless, the results indicate, that fermentation with different supplementation with a proper combination of a S. thermophilus , L. casei and B. breve resulted in a fermented milk with the character of a "yoghurt mild", but without the mixture of the classical yoghurt microflora.

Conclusion

Production of fermented milk with mixed culture of L. casei, B. breve and S. thermophilus (CBT) supplemented with different additives was carried out. Fortified fermented milk showed full yoghurt flavor, and also body and texture was firm, more compact, and quite better than control. pH was nearly stable during the storage at 5[degrees] C. In addition the high total viable counts of S. thermophilus , L. casei and B.breve (CBT) starter culture were an important advantage of the production of fortified probiotic fermented milk. Nevertheless, the results indicate, that fermentation with different supplementation with a proper combination of a S. thermophilus , L. casei and B. breve resulted in a fermented milk with the character of a "yoghurt mild", but without the mixture of the classical yoghurt This result showed that peptides and amino acids have improved the viability of probiotic bacteria, especially bifidobacteria, which have been reported to be weakly proteolytic.

Acknowledgments

Our thanks are due to Dr. Chr. Lorenzen BfEL, Kiel, Germany for provid us by GMP.

Referances

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H. A. EL-Demerdash (1) and M.Al.Otiabi (2)

(1) Food and Dairy Science Dept., Fac. of Environ Agric Sci. Suez Canal Univ., EL- Arish, Egypt.

(2) Food and Nutrition Sciences Dept. Fac. of Agric and Food Sci KFU, KSA hassanam7@hotmail.com
Table 1: Synersis in fermented milk fortified with various
ingredients during refrigerated storage for 21 day.

          Drain method          Centrifuge method
Samples   ml/100g)              (ml/100g)
          Storage time (days)

          0    7    14   21     0    7    14   21
Control   33   37   40   45     53   58   64   67
T1        32   35   38   40     52   56   61   66
T2        20   22   25   30     40   45   49   54
T3        17   19   21   23     34   36   37   40
T4        18   21   24   26     36   39   42   44

Table 2: Rheological parameters of fermented milk fortified with
various ingredients during storage for 21 days. See table (1)
for samples designation.

          Viscosity (mPa.S)           Flow index
Samples   Time (days)
          0      7      14     21     0      7      14     21
Control   2160   2480   2760   2894   0.61   0.57   0.52   0.49
T1        2154   2511   2754   2897   0.59   0.56   0.52   0.48
T2        2356   2720   3176   3245   0.44   0.39   0.34   0.31
T3        2714   3299   3624   3814   0.28   0.25   0.22   0.18
T4        2578   3118   3290   3465   0.39   0.34   0.28   0.24

Table 3: Carbohydrate analysis of the fermented milk inculcated by
(CBT) starter culture and fortified with different ingredients during
storage period. See Table (1) for samples designation.

           Lactose                 Glucose
Samples    (%)
           Storage period (days)
           1          7          14         21         1
Control    4.49       3.74       3.11       3.07       0.14
T1         4.49       3.74       3.11       3.07       0.14
T2         4.50       3.75       3.05       2.59       0.18
T3         4.69       3.93       3.21       2.67       0.20
T4         5.08       4.34       3.66       3.01       0.28

                      Galactose
Samples

           7          14         21         1          7
Control    0.11       0.1        0.07       1.13       1.15
T1         0.11       0.1        0.07       1.13       1.15
T2         0.12       0.11       0.06       1.11       1.13
T3         0.16       0.12       0.05       1.10       1.15
T4         0.15       0.14       0.03       1.14       1.14

             Galactose
Samples

           14         21
Control    1.21       1.24
T1         1.21       1.24
T2         1.20       1.26
T3         1.19       1.27
T4         1.19       1.31

Table 4: Organoleptic characteristics of fermented milk fortified
with various ingredients during refrigerated storage for 21 days.
See Table (1) for samples designation.

Treatments
Storage Control           T1    T2   T3   T4   Periods
Flavour (10)
1day                        8    8    8    8    9
7days                       8    7    7    9    9
14days                      7    7    6    8    8
21days                      6    6    6    7    8
Body & Texture (5)
1day                        4    4    5    5    5
7days                       3    3    4    4    4
14days                      3    3    3    4    4
21days                      2    2    3    4    4
Color and appearance (5)
1day                        4    4    5    5    5
7days                       3    3    4    4    4
14days                      3    3    4    4    4
21days                      2    2    3    4    4
Total score (20 points)
1day                       16   16   18   18   18
7days                      14   13   15   18   17
14days                     12   13   13   16   16
21days                     10   10   12   15   16
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Author:Demerdash, H.A. El-; Otiabi, M.Al.
Publication:International Journal of Biotechnology & Biochemistry
Date:May 1, 2008
Words:5246
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