supplemented with LAB was 2.2, 2.3 and 0.3 ml 1 M NaOH ∙ 100 g-1, respectively.

0.7 ml 1 M NaOH ∙ 100 g-1, were noticed in raisins coated with dark couverture.

LAB increased by 0.1 and 0.6 ml 1 M NaOH ∙ 100 g-1, respectively.

depended on storage temperature.

**Dark + LAB Milk + LAB White**

**Storage time**

30°C for 3 months.

couverture.

**Table 25.** Water activity in chocolate coated raisins, received with the use of different types of couverture **supplemented**, and as a comparison **non-supplemented** with LAB, stored at temperatures of 4, 18 and 30°C for 3 months.

Raisins coated with dark and milk couverture without LAB addition showed similar water activity values (for whole chocolate coated raisins). Slightly lower value of aw had raisins coated with white couverture. Supplementation of couvertures with lactic acid bacteria only very slightly increased the values of aw in final products, obtained with the use of dark and milk couvertures. More noticeable increase of aw – from 0.389 to 0.414 was observed for raisins coated with white couverture.

During storage of raisins coated with all types of couverture supplemented with LAB at refrigeration and room temperatures water activity increased (whole raisins in chocolate). Only at higher storage temperature of 30°C water activity was decreasing for 2 months of storage to finally increase during third month. Similar changes of aw during storage were observed for raisins coated with non-supplemented couverture. A difference was noticed for aw changes of chocolate coated raisins stored at 30°C, in which during first month of storage aw decreased, and during following months of storage rose to values higher than in initial samples (directly after production).

Water activity in crushed products was generally higher comparing to the values of this parameter analyzed in a whole product. Comparing water activity values in whole and crushed raisins coated with chocolate, obtained with the use of supplemented with LAB and non-supplemented couvertures – dark, milk and white, directly after production and during 3 months of storage at temperatures of 4, 18 and 30°C it can be concluded that they kept under the value of 0.6. Due to that fact, it is probable that during the whole time of storage no bacterial activity in both, supplemented and non-supplemented with LAB, will be maintained.

#### *Total acidity in chocolate coated raisins*

Total acidity changes of chocolate coated raisins during 3 months of storage in various temperatures is presented in Table 26.

**2 months**

**3 months**

crushed **0.499** ±

whole **0.406** ±

crushed **0.495** ±

whole **0.433** ±

crushed **0.456** ±

raisins coated with white couverture.

samples (directly after production).

*Total acidity in chocolate coated raisins*

temperatures is presented in Table 26.

maintained.

of 4, 18 and 30°C for 3 months.

0.011

0.003

0.007

0.002

0.004

**0.463** ± 0.003

**0.295** ± 0.004

**0.451** ± 0.004

**0.478** ± 0.002

**0.487** ± 0.007

**Table 25.** Water activity in chocolate coated raisins, received with the use of different types of couverture **supplemented**, and as a comparison **non-supplemented** with LAB, stored at temperatures

Raisins coated with dark and milk couverture without LAB addition showed similar water activity values (for whole chocolate coated raisins). Slightly lower value of aw had raisins coated with white couverture. Supplementation of couvertures with lactic acid bacteria only very slightly increased the values of aw in final products, obtained with the use of dark and milk couvertures. More noticeable increase of aw – from 0.389 to 0.414 was observed for

During storage of raisins coated with all types of couverture supplemented with LAB at refrigeration and room temperatures water activity increased (whole raisins in chocolate). Only at higher storage temperature of 30°C water activity was decreasing for 2 months of storage to finally increase during third month. Similar changes of aw during storage were observed for raisins coated with non-supplemented couverture. A difference was noticed for aw changes of chocolate coated raisins stored at 30°C, in which during first month of storage aw decreased, and during following months of storage rose to values higher than in initial

Water activity in crushed products was generally higher comparing to the values of this parameter analyzed in a whole product. Comparing water activity values in whole and crushed raisins coated with chocolate, obtained with the use of supplemented with LAB and non-supplemented couvertures – dark, milk and white, directly after production and during 3 months of storage at temperatures of 4, 18 and 30°C it can be concluded that they kept under the value of 0.6. Due to that fact, it is probable that during the whole time of storage no bacterial activity in both, supplemented and non-supplemented with LAB, will be

Total acidity changes of chocolate coated raisins during 3 months of storage in various

0.007 0.009 0.001 0.001 0.004

**0.495** ± 0.014

**0.476** ± 0.050

**0.483** ± 0.020

**0.472** ± 0.070

**0.480** ± 0.009

**0.472** ± 0.003

**0.481** ± 0.005

**0.490** ± 0.005

**0.457** ± 0.002

**0.469** ± 0.007

**0.513** ± 0.002

**0.493** ± 0.001

**0.474** ± 0.011

**0.462** ± 0.001

**0.469** ± 0.009

**0.477** ± 0.006

**0.404** ± 0.004

**0.461** ± 0.002

**0.469** ± 0.002

**0.488** ± 0.005

> **Table 26.** Total acidity in chocolate coated raisins, received with the use of different types of couverture **supplemented**, and as a comparison **non-supplemented** with LAB, stored at temperatures of 4, 18 and 30°C for 3 months.

> Directly after obtaining the highest total acidity, amounting 17.7 ml 1 M NaOH ∙ 100 g-1, had raisins coated with dark couverture non-supplemented with LAB, and the lowest, amounting 13.6 ml 1 M NaOH ∙ 100 g-1, was observed in raisins coated with white couverture.

> Supplementation of dark, milk and white couvertures with bacteria from *Lactobacillus* species didn't influence significantly the total acidity of products after production. The biggest changes of this parameter after supplementation of couverture with LAB, namely by 0.7 ml 1 M NaOH ∙ 100 g-1, were noticed in raisins coated with dark couverture.

> In raisins coated with white and milk couvertures total acidity after supplementation with LAB increased by 0.1 and 0.6 ml 1 M NaOH ∙ 100 g-1, respectively.

> During storage of chocolate coated raisins only slight decrease of total acidity was observed. With an exception in product containing milk couverture non-supplemented with LAB, in which total acidity increased by 1 ml 1 M NaOH ∙ 100 g-1 after 3 months of storage at a temperature of 30°C. The magnitude of total acidity changes in chocolate coated raisins depended on storage temperature.

> In raisins coated with dark, white and milk couverture supplemented with LAB total acidity decrease during storage. The biggest decrease of this parameter was noticed in chocolate coated raisins stored at 30°C and in products in dark, milk and white couverture supplemented with LAB was 2.2, 2.3 and 0.3 ml 1 M NaOH ∙ 100 g-1, respectively.

#### *Analysis of fat quality in a coating of chocolate coated raisins by DSC method*

In obtained chocolate coated raisins, analyses of changes occurring in fat from a couverture (which is a coating of the product), by differential scanning calorimetry method were performed. These changes are presented in tables 30 and 31. Exemplary thermograms of fats from dark couverture supplemented and non-supplemented with LAB, which are a coating of chocolate coated raisins, stored during 3 months period at a temperature of 18°C are presented in Figures 8 and 9, respectively.

Probiotic Confectionery Products – Preparation and Properties 297

During storage of chocolate coated raisins melting enthalpy value of fat from coatings of dark and white chocolate coated raisins, increased regardless of storage conditions. It can be caused by crystallization of previously present sources of crystallization or by increasing the

**control** 30.157 35.51 27.559 34.34 34.93 34.25 **Stored at 4oC 1 month** 31.568 35.90 25.400 33.94 22.026 34.61

3.744+20.129

**Stored at 18oC 1 month** 36.765 34.54 26.622 33.57 32.684 34.86

1.580+26.59

**Stored at 30oC**

5.225+18.834

18.764 5.244+13.519

24.259 6.087+18.171

**Table 28.** Enthalpy (ΔH) and melting temperature (Tm) of fat from coatings of chocolate coated raisins obtained with the use of different types of couvertures **non-supplemented** with LAB, stored at

Temperature and storage time of chocolate coated raisins had an influence on changes of polymorphic forms of fat from couverture coating products. In coatings without LAB addition, bigger tendency to two polymorphic forms creation was observed, mainly in raisins stored at temperatures of 18 and 30°C. The range of maximal melting temperatures of first polymorphic form of fat from couverture from chocolate coated raisins nonsupplemented with LAB was from 26.80 to 32.30°C. Whereas for second polymorphic form it ranged from 33.69 to 35.95°C. Range of melting temperatures of fat from couverture from chocolate coated raisins supplemented with LAB, for first polymorphic form was from 29.57 to 33.01°C, and for second form, from 34.10 to 38.82°C.

**Storage time**

**"0"**

**2 months** 33.064

**3 months** 38.068

**2 months** 29.962

**3 months** 33.30

0.911+32.153

25.119+12.917

0.741+29.22

4.558+28.743

temperatures of 4, 18 and 30°C during 3 months.

**3 months** 37.718 34.25 23.874

**2 months** 31.847 36.20 28.173

**1 month** 36.373 35.35 24.059

Tm1= 28.64

Tm1= 32.32

Tm1=29.14 Tm2=35.94

Tm=31.04 Tm=35.95

**Table 27.** Enthalpy (ΔH) and melting temperature (Tm) of fat from coatings of chocolate coated raisins obtained with the use of different types of couvertures **supplemented** with LAB, stored at temperatures of 4, 18 and 30°C during 3 months.

In samples of chocolate coated raisins directly after production the value of melting enthalpy of fat extracted from product coating, in all types of couvertures supplemented with lactic acid bacteria was lower than in analogous products coated with nonsupplemented couverture. This phenomena can be explained by the fact, that LAB preparation influenced fat crystallization, namely in supplemented couverture more liquid phase of fat was present than in analogous products without LAB.

During storage of chocolate coated raisins melting enthalpy value of fat from coatings of dark and white chocolate coated raisins, increased regardless of storage conditions. It can be caused by crystallization of previously present sources of crystallization or by increasing the area of already existing fat crystals.

296 Probiotics

**Storage time**

**3 months** 29.820

**2 months** 32.475

**3 months** 36.578

of 4, 18 and 30°C during 3 months.

16.564 + 13.255

2.440 + 30.034

22.555+14.022

*Analysis of fat quality in a coating of chocolate coated raisins by DSC method*

presented in Figures 8 and 9, respectively.

In obtained chocolate coated raisins, analyses of changes occurring in fat from a couverture (which is a coating of the product), by differential scanning calorimetry method were performed. These changes are presented in tables 30 and 31. Exemplary thermograms of fats from dark couverture supplemented and non-supplemented with LAB, which are a coating of chocolate coated raisins, stored during 3 months period at a temperature of 18°C are

**Control "0"** 23.014 34.30 22.906 34.30 24.020 34.20 **Stored at 4oC 1 month** 35.480 34.78 16.530 34.51 22.357 34.47

**3 months** 25.261 34.51 25.218 34.03 33.653 34.15 **Stored at 18oC 1 month** 35.742 34.54 24.230 33.94 17.124 34.73 **2 months** 34.312 34.77 22.906 33.46 25.591 34.00

**Stored at 30oC 1 month** 34.000 36.45 16.353 34.96 29.053 35.08

> 18.786 7.561+11.229

> 24.102 5.988+18.113

**Table 27.** Enthalpy (ΔH) and melting temperature (Tm) of fat from coatings of chocolate coated raisins obtained with the use of different types of couvertures **supplemented** with LAB, stored at temperatures

In samples of chocolate coated raisins directly after production the value of melting enthalpy of fat extracted from product coating, in all types of couvertures supplemented with lactic acid bacteria was lower than in analogous products coated with nonsupplemented couverture.

**Enthalpy and melting temperature of fat from coatings of chocolate coated raisins: Dark + LAB Milk + LAB White + LAB**  ΔH (J/g) Tm (°C) ΔH (J/g) Tm (°C) ΔH (J/g) Tm (°C)

Tm2=34.10 34.744 32.48 33.698 34.74

Tm1=31.48

Tm1=29.67 Tm2=34.42 4.487 + 23.009

Tm2=35.12 23.137 35.60

35.622 8.977+26.645 Tm1=30.58 Tm2=34.54

Tm1=31.11 Tm2=35.34

**Table 28.** Enthalpy (ΔH) and melting temperature (Tm) of fat from coatings of chocolate coated raisins obtained with the use of different types of couvertures **non-supplemented** with LAB, stored at temperatures of 4, 18 and 30°C during 3 months.

Temperature and storage time of chocolate coated raisins had an influence on changes of polymorphic forms of fat from couverture coating products. In coatings without LAB addition, bigger tendency to two polymorphic forms creation was observed, mainly in raisins stored at temperatures of 18 and 30°C. The range of maximal melting temperatures of first polymorphic form of fat from couverture from chocolate coated raisins nonsupplemented with LAB was from 26.80 to 32.30°C. Whereas for second polymorphic form it ranged from 33.69 to 35.95°C. Range of melting temperatures of fat from couverture from chocolate coated raisins supplemented with LAB, for first polymorphic form was from 29.57 to 33.01°C, and for second form, from 34.10 to 38.82°C. Range of melting temperatures of first and second polymorphic forms of fat was similar in raisins coated with both, supplemented and non-supplemented couvertures. At a temperature of 30°C in dark and milk couvertures both, supplemented and non-supplemented, appearance of second polymorphic form of fat was observed after 2 months of storage. In white couverture appearance of second polymorphic form was noticed after 3 month storage of product. In can concluded that lack of cocoa liquor and bigger content of milk in a white couverture delayed polymorphic changes of fats in this couverture.

Probiotic Confectionery Products – Preparation and Properties 299

Dark and milk chocolate coated raisins supplemented with lactic acid bacteria became harder. On the other hand raisins in white chocolate after supplementation with LAB

During storage, from all supplemented products, raisins coated with white couverture became the hardest. During storage of all studied chocolate coated raisins (with and without LAB addition) at temperatures of 4 and 18°C hardness gradually decreased, which can be associated with water diffusion. During storage of supplemented and non-supplemented chocolate coated raisins at temperature of 30°C hardness initially rose (drying of surface), next it decreased (water diffusion from raisin to coating and from environment into product), and finally to increase after third month. Additionally hardness of supplemented products was higher than hardness analyzed directly after production. With an exception of raisins coated with white chocolate, in which hardness was significantly lower than in fresh

> **Force (kg) required to cut chocolate coated raisins: Dark + LAB Milk + LAB White + LAB Dark Milk White**

**Control "0"** 3.002 2.949 3.781 2.777 2.850 3.861 **Stored at 4oC 1 month** 3.005 3.125 3.531 2.461 2.596 2.344 **2 months** 2.821 2.731 2.424 2.247 2.492 2.067 **3 months** 2.078 2.068 2.329 2.227 2.271 1.838 **Stored at 18oC 1 month** 2.712 3.007 3.319 2.023 3.110 1.898 **2 months** 2.512 2.597 2.785 1.772 2.316 1.723 **3 months** 2.125 2.136 2.015 2.180 2.527 2.037 **Stored at 30oC 1 month** 3.010 3.154 3.337 2.832 3.480 2.156 **2 months** 2.786 2.727 2.751 2.314 3.321 2.139 **3 months** 3.068 3.404 3.608 2.649 2.641 2.413 **Table 29.** Force required to cut chocolate coated raisins received with the use of different types of couverture **supplemented**, and as a comparison **non-supplemented** with LAB, stored at temperatures

In received raisins coated with couverture supplemented with LAB organoleptic analysis was performed, according to a 5-point scale, and it was compared to products obtained with

The highest note in a 5-point scale received raisins coated with white chocolate and then with milk couverture. Lowest ratings (below 4 points) received raisins coated with dark chocolate both, fresh and during the whole storage period, regardless of LAB supplementation. To high grade of raisins coated with white couverture was caused by their

softened.

products.

**Storage time**

of 4, 18 and 30°C for 3 months.

*Organoleptic evaluation of chocolate coated raisins*

non-supplemented courevture (Table 30).

**Figure 8.** Thermogram of fat from coatings of chocolate coated raisins obtained from dark couverture **supplemented** with LAB stored at a temperature of 18°C during 3 months.

**Figure 9.** Thermogram of fat from coatings of chocolate coated raisins obtained from dark couverture **non-supplemented** with LAB stored at a temperature of 18°C during 3 months.

#### *Texture of chocolate coated raisins*

In Table 29 results of cutting test of chocolate coated raisins are presented.

Dark and milk chocolate coated raisins supplemented with lactic acid bacteria became harder. On the other hand raisins in white chocolate after supplementation with LAB softened.

During storage, from all supplemented products, raisins coated with white couverture became the hardest. During storage of all studied chocolate coated raisins (with and without LAB addition) at temperatures of 4 and 18°C hardness gradually decreased, which can be associated with water diffusion. During storage of supplemented and non-supplemented chocolate coated raisins at temperature of 30°C hardness initially rose (drying of surface), next it decreased (water diffusion from raisin to coating and from environment into product), and finally to increase after third month. Additionally hardness of supplemented products was higher than hardness analyzed directly after production. With an exception of raisins coated with white chocolate, in which hardness was significantly lower than in fresh products.

**Table 29.** Force required to cut chocolate coated raisins received with the use of different types of couverture **supplemented**, and as a comparison **non-supplemented** with LAB, stored at temperatures of 4, 18 and 30°C for 3 months.

#### *Organoleptic evaluation of chocolate coated raisins*

298 Probiotics

first and second polymorphic forms of fat was similar in raisins coated with both, supplemented and non-supplemented couvertures. At a temperature of 30°C in dark and milk couvertures both, supplemented and non-supplemented, appearance of second polymorphic form of fat was observed after 2 months of storage. In white couverture appearance of second polymorphic form was noticed after 3 month storage of product. In can concluded that lack of cocoa liquor and bigger content of milk in a white couverture

**2 months**

**25.5 28.0 30.5 33.0 35.5 38.0 40.5 43.0 45.5**

**25.5 28.0 30.5 33.0 35.5 38.0 40.5 43.0 45.5**

**Figure 9.** Thermogram of fat from coatings of chocolate coated raisins obtained from dark couverture

**2 months**

**non-supplemented** with LAB stored at a temperature of 18°C during 3 months.

In Table 29 results of cutting test of chocolate coated raisins are presented.

**supplemented** with LAB stored at a temperature of 18°C during 3 months.

**Figure 8.** Thermogram of fat from coatings of chocolate coated raisins obtained from dark couverture

**Control**

**1month**

**3 months**

**Temperature/ °C**

**3 months**

**Control**

**Temperature/ °C**

**LAB**

delayed polymorphic changes of fats in this couverture.

**Heat Flow/ mW**

**-65 -63 -61 -59 -57 -55 -53 -51 -49 -47 Exo**

**Heat Flow/ mW**

**1 month**

**-65.0 -62.5 -60.0 -57.5 -55.0 -52.5 -50.0 -47.5 -45.0 -42.5 Exo**

*Texture of chocolate coated raisins*

In received raisins coated with couverture supplemented with LAB organoleptic analysis was performed, according to a 5-point scale, and it was compared to products obtained with non-supplemented courevture (Table 30).

The highest note in a 5-point scale received raisins coated with white chocolate and then with milk couverture. Lowest ratings (below 4 points) received raisins coated with dark chocolate both, fresh and during the whole storage period, regardless of LAB supplementation. To high grade of raisins coated with white couverture was caused by their delicate, gentle taste, and soft, elastic consistency. Addition of lactic acid bacteria to couvertures coating raisins didn't influence significantly sensory properties of products, which is favorable from the point of view of a consumer, who highly appreciates sensory quality of chocolate. However, although in first month of storage of chocolate coated raisins stored at all temperatures organoleptic rating didn't change, in latter months this parameter degraded, especially when stored at 30°C. It was caused by the changes occurring in products during storage. Most noticeably these changes were observed in chocolate coated raisins stored at 30°C. They included changes of taste, caused by modifications of fat in a coating, an increase of dry mass content in cores (raisins), increase of hardness of raisins coated with dark and milk chocolates, connected to an increase of dry mass content in chocolate couvertures and surfaces of products, which became less shiny with time.

Probiotic Confectionery Products – Preparation and Properties 301

**Storage temperature**

4°C 18°C 30°C

**Viability of bacteria (%)**

**Storage temperature**

The amount of live bacterial cells in the product

(CFU .

4°C 18°C 30°C

80 g-1)

The biggest viability of *Lactobacillus* bacteria in products after 3 months of storage was observed when stored at a refrigeration temperature (4°C). The highest viability was observed in raisins coated with dark (88.9%) and white (88.0%) chocolate, slightly lower was noticed in raisins coated with milk chocolate (86.5%) (Table 31). In products stored at a temperature of 18°C amount of live bacterial cells was lower by two orders of magnitude,

decrease in an amount of live cells was observed, even just after one month of storage. The biggest drop in LAB viability was observed in raisins coated with white chocolate, to 58.6%, next in raisins coated with milk chocolate, to 59.7%, and finally in raisins coated with dark

Raisins coated with milk chocolate **88.0**  2.3 **73.1**  3.0 **59.7**  3.9

Raisins coated with dark chocolate **88.9**  3.0 **72.7**  3.8 **61.3**  4.0

Raisins coated with white chocolate **86.5**  2.7 **66.3**  2.0 **58.6**  4.0

chocolate 3.4108 2.5107 2.5106

chocolate 3.8108 2.3107 3.2106

chocolate 2.7108 8.0106 2.1106

**Table 32.** The amount of live bacterial cells of *Lactobacillus* species in chocolate coated raisins, with a

**Table 31.** Viability of *Lactobacillus* bacteria in chocolate coated raisins after 3 months of storage.

g-1. When products were stored at a stress temperature (30°C) a severe

*Viability of LAB in a product*

amounting 105 CFU .

**Sample**

**Sample**

Raisins coated with milk

Raisins coated with dark

Raisins coated with white

weight of 80g, after 3 months of storage.

chocolate viability lowered to 61.3%.

Organoleptic ratings of raisins coated with dark, milk and white chocolates stored at temperatures of 4 and 18°C were practically identical in a first month of storage (differences of 0.0 – 0.1 points) comparing to fresh product, they were slightly different after second month (by 0.0 – 0.2 points) and third month (0.0 – 0.1 points) of storage. In case of 3 month storage period of non-supplemented products, differences in organoleptic evaluation between fresh product and product stored for a 3 month period were more noticeable and reached 0.7 points. Considering similar organoleptic evaluation of raisins coated with chocolate stored at 4 and 18°C it can be concluded, that examined raisins coated with chocolate don't have to be kept at refrigeration conditions and can be stored at a store shelf as well, where they can easily be found by a consumer next to analogous traditional products.

**Table 30.** Organoleptic evaluation of chocolate coated raisins received with the use of different types of couverture **supplemented**, and as a comparison **non-supplemented** with LAB, stored at temperatures of 4, 18 and 30°C for 3 months.

#### *Viability of LAB in a product*

300 Probiotics

products.

**Storage time**

of 4, 18 and 30°C for 3 months.

delicate, gentle taste, and soft, elastic consistency. Addition of lactic acid bacteria to couvertures coating raisins didn't influence significantly sensory properties of products, which is favorable from the point of view of a consumer, who highly appreciates sensory quality of chocolate. However, although in first month of storage of chocolate coated raisins stored at all temperatures organoleptic rating didn't change, in latter months this parameter degraded, especially when stored at 30°C. It was caused by the changes occurring in products during storage. Most noticeably these changes were observed in chocolate coated raisins stored at 30°C. They included changes of taste, caused by modifications of fat in a coating, an increase of dry mass content in cores (raisins), increase of hardness of raisins coated with dark and milk chocolates, connected to an increase of dry mass content in

chocolate couvertures and surfaces of products, which became less shiny with time.

**Dark + LAB Milk + LAB White +**

Organoleptic ratings of raisins coated with dark, milk and white chocolates stored at temperatures of 4 and 18°C were practically identical in a first month of storage (differences of 0.0 – 0.1 points) comparing to fresh product, they were slightly different after second month (by 0.0 – 0.2 points) and third month (0.0 – 0.1 points) of storage. In case of 3 month storage period of non-supplemented products, differences in organoleptic evaluation between fresh product and product stored for a 3 month period were more noticeable and reached 0.7 points. Considering similar organoleptic evaluation of raisins coated with chocolate stored at 4 and 18°C it can be concluded, that examined raisins coated with chocolate don't have to be kept at refrigeration conditions and can be stored at a store shelf as well, where they can easily be found by a consumer next to analogous traditional

**Grades (points) of chocolate coated raisins:**

**Control "0" 4.0** ±0.1 **4.2** ± 0.1 **4.7** ± 0.2 **3.9** ± 0.0 **4.0** ± 0.1 **4.6** ± 0.1 **Stored at4oC 1 month 3.7** ± 0.1 **4.1** ± 0.1 **4.6** ± 0.3 **3.9** ± 0.2 **3.9** ± 0.2 **4.5** ± 0.3 **2 months 3.6** ± 0.2 **4.1** ± 0.1 **4.6** ± 0.1 **3.8** ± 0.2 **3.9** ± 0.1 **4.6** ± 0.1 **3 months 3.1** ± 0.0 **3.9** ± 0.1 **4.5** ± 0.2 **3.6** ± 0.1 **3.8** ± 0.2 **4.5** ± 0.2 **Stored at18oC 1 month 3.8** ± 0.2 **4.0** ± 0.2 **4.6** ± 0.2 **3.8** ± 0.2 **3.9** ± 0.1 **4.5** ± 0.3 **2 months 3.7** ± 0.2 **4.0** ± 0.1 **4.5** ± 0.2 **3.7** ± 0.2 **3.8** ± 0.2 **4.3** ± 0.2 **3 months 3.0** ± 0.1 **3.7** ± 0.2 **4.4** ± 0.3 **3.4** ± 0.1 **3.1** ± 0.3 **4.0** ± 0.2 **Stored at30oC 1 month 3.9** ± 0.2 **4.1** ± 0.2 **4.6** ± 0.2 **3.9** ± 0.1 **3.8** ± 0.2 **4.5** ± 0.1 **2 months 3.7** ± 0.2 **3.9** ± 0.3 **4.4** ± 0.2 **3.4** ± 0.2 **3.7** ± 0.1 **4.2** ± 0.2 **3 months 2.8** ± 0.3 **3.6** ± 0.2 **3.5** ± 0.2 **2.9** ± 0.2 **3.0** ± 0.1 **3.4** ± 0.2 **Table 30.** Organoleptic evaluation of chocolate coated raisins received with the use of different types of couverture **supplemented**, and as a comparison **non-supplemented** with LAB, stored at temperatures

**LAB Dark Milk White**

The biggest viability of *Lactobacillus* bacteria in products after 3 months of storage was observed when stored at a refrigeration temperature (4°C). The highest viability was observed in raisins coated with dark (88.9%) and white (88.0%) chocolate, slightly lower was noticed in raisins coated with milk chocolate (86.5%) (Table 31). In products stored at a temperature of 18°C amount of live bacterial cells was lower by two orders of magnitude, amounting 105 CFU . g-1. When products were stored at a stress temperature (30°C) a severe decrease in an amount of live cells was observed, even just after one month of storage. The biggest drop in LAB viability was observed in raisins coated with white chocolate, to 58.6%, next in raisins coated with milk chocolate, to 59.7%, and finally in raisins coated with dark chocolate viability lowered to 61.3%.

**Table 31.** Viability of *Lactobacillus* bacteria in chocolate coated raisins after 3 months of storage.

**Table 32.** The amount of live bacterial cells of *Lactobacillus* species in chocolate coated raisins, with a weight of 80g, after 3 months of storage.

The worst *Lactobacillus* bacteria viability, at all storage temperatures, was observed in raisins coated with white couverture. Storage of raisins coated with dark, white and milk couvertures supplemented with *Lactobacillus* bacteria, at a temperature of 4°C provides a maintenance of probiotic properties of these products. Temperature of 18°C, in case of raisins coated with dark and milk couvertures, also prevents them from loosing probiotic properties during storage. Storage of those products in this temperature allows to maintain high lactic bacteria viability during the whole storage period, namely 3 months.

Probiotic Confectionery Products – Preparation and Properties 303

became softer. During storage at temperatures of 4 and 18°C raisins coated with dark, milk and white couvertures supplemented with LAB gradual decrease in hardness was observed. After 2 months of storage products were softer than when

Viability of lactic acid bacteria in some confectionery products appears to be unexpectedly high. It is caused by low moisture content in products mentioned in this chapter, as well as reqiured water activity (below 0.6), high concentration of carbohydrates, mainly saccharose and limited access of oxygen. However, viability depends mainly on recipe of product (mainly the type of fat), technological processes used for obtaining products and the time of

Many solutions for application of lactic acid bacteria to confectionery, pastry and other

Application of bacteria in a form of preserved preparation, in which live cells are put in a state of anabiosis, allows to maintain high viability of LAB in confectionery products during storage. LAB addition to confectionery products - a type of food often consumed by kids and youth, allows to enrich the diet of this group of consumers with probiotic products with taste similar to traditional products, which are also ready for distribution and sale analogous to products without addition of bacterial preparations, not requiring refrigeration

Dorota Żyżelewicz, Ilona Motyl, Ewa Nebesny, Grażyna Budryn, Wiesława Krysiak,

Authors wish to thank Polish Ministry of Science and High Education for financial support of research and development project No. R12 018 01 about: "Semi-products and Products Suplemented with Viable Lactic Acid Bacteria" in which presented studies were performed.

*University of Technology, Faculty of Biotechnology and Food Sciences, Lodz, Poland*

kinds of products, cited in this chapter, is the subject of patent protection.

they were fresh.

couverture.

**7. Conclusion**

**Author details**

**Acknowledgement**

storage period remained at a functional level.

these processes, and finally storage conditions.

temperatures and hence being always "within reach".

Justyna Rosicka-Kaczmarek and Zdzisława Libudzisz

Consuming a package of chocolate coated raisins, with a weight of 80 g, stored at 4°C provides a consumer with 108 CFU of probiotic bacteria. The same package of raisins coated with dark and milk chocolate stored at a temperature of 18°C contains 2.3107 CFU . 80 g-1 and 2.5107 CFU . 80 g-1, respectively, while raisins coated with white chocolate an amount of 8.02106 CFU . 80 g-1 of final product (Table 32). After storage of chocolate coated raisins at 30°C consumed amount of lactic acid bacteria would amount to a level of 106 CFU . 80 g-1 of final product, and would be below recommended level (107 CFU . g-1) for functional food.

#### **6. Summary**

Proposed technology enables to introduce to dark, white and milk couvertures, live cultures of lactic acid bacteria (as a lyophilisate) and to use them for obtaining raisins coated with chocolate, characterized by soft consistency.

Results of research and development project indicated what follows:

became softer. During storage at temperatures of 4 and 18°C raisins coated with dark, milk and white couvertures supplemented with LAB gradual decrease in hardness was observed. After 2 months of storage products were softer than when they were fresh.

## **7. Conclusion**

302 Probiotics

months.

.

80 g-1 and 2.5107 CFU .

amount of 8.02106 CFU .

chocolate, characterized by soft consistency.

slight lowering of this parameter.

functional food.

**6. Summary**

slightly.

form.

The worst *Lactobacillus* bacteria viability, at all storage temperatures, was observed in raisins coated with white couverture. Storage of raisins coated with dark, white and milk couvertures supplemented with *Lactobacillus* bacteria, at a temperature of 4°C provides a maintenance of probiotic properties of these products. Temperature of 18°C, in case of raisins coated with dark and milk couvertures, also prevents them from loosing probiotic properties during storage. Storage of those products in this temperature allows to maintain high lactic bacteria viability during the whole storage period, namely 3

Consuming a package of chocolate coated raisins, with a weight of 80 g, stored at 4°C provides a consumer with 108 CFU of probiotic bacteria. The same package of raisins coated with dark and milk chocolate stored at a temperature of 18°C contains 2.3107 CFU .

raisins at 30°C consumed amount of lactic acid bacteria would amount to a level of 106 CFU

Proposed technology enables to introduce to dark, white and milk couvertures, live cultures of lactic acid bacteria (as a lyophilisate) and to use them for obtaining raisins coated with

80 g-1 of final product, and would be below recommended level (107 CFU .

Results of research and development project indicated what follows:

increase of aw was noticed for raisins coated with white couverture.

80 g-1, respectively, while raisins coated with white chocolate an

80 g-1 of final product (Table 32). After storage of chocolate coated

g-1) for

Viability of lactic acid bacteria in some confectionery products appears to be unexpectedly high. It is caused by low moisture content in products mentioned in this chapter, as well as reqiured water activity (below 0.6), high concentration of carbohydrates, mainly saccharose and limited access of oxygen. However, viability depends mainly on recipe of product (mainly the type of fat), technological processes used for obtaining products and the time of these processes, and finally storage conditions.

Many solutions for application of lactic acid bacteria to confectionery, pastry and other kinds of products, cited in this chapter, is the subject of patent protection.

Application of bacteria in a form of preserved preparation, in which live cells are put in a state of anabiosis, allows to maintain high viability of LAB in confectionery products during storage. LAB addition to confectionery products - a type of food often consumed by kids and youth, allows to enrich the diet of this group of consumers with probiotic products with taste similar to traditional products, which are also ready for distribution and sale analogous to products without addition of bacterial preparations, not requiring refrigeration temperatures and hence being always "within reach".

## **Author details**

Dorota Żyżelewicz, Ilona Motyl, Ewa Nebesny, Grażyna Budryn, Wiesława Krysiak, Justyna Rosicka-Kaczmarek and Zdzisława Libudzisz *University of Technology, Faculty of Biotechnology and Food Sciences, Lodz, Poland*

## **Acknowledgement**

Authors wish to thank Polish Ministry of Science and High Education for financial support of research and development project No. R12 018 01 about: "Semi-products and Products Suplemented with Viable Lactic Acid Bacteria" in which presented studies were performed. We also would like to acknowledge help received from companies: ZPT "Kruszwica" S.A. (Kruszwica, Poland) and AARHUSKARLSHAMN SWEDEN AB (Karlshamn, Sweden) for transferring a portion of fats used for obtaining nutty fatty mass and wafer creams.

Probiotic Confectionery Products – Preparation and Properties 305

[15] Żyżelewicz D, Nebesny E, Motyl I, Libudzisz Z. Effect of milk chocolate supplementation with lyophilized *Lactobacillus* cells on its attributes. Czech Journal of

[16] Nebesny E, Żyżelewicz D, Motyl I, Libudzisz Z. Dark chocolates supplemented with *Lactobacillus* strains. European Food Research and Technology 2007;225(5) 33-42. [17] Cukrowska B, Ceregra A, Rosiak I, Libudzisz Z, Motyl I, Klewicka E. Effect of Oral Dosage of Probiotic Bacteria *Lactobacillus* on Composition of Gut Ecosystem, Immune System and Course of the Food Allergy in Infants. Grant's rapport No KBN PB

[18] Sokmen A, Gunes G. Influence of some bulk sweeteners on rheological properties of

[19] Chevalley J. An adaptation of the Casson equation for the rheology of chocolate. Journal

[20] Steiner EH. A new rheological relationship to express the flow properties of melted

[21] Foubert I, Vanrolleghem PA, Dewettinck K. A differential scanning calorimetry method to determine the isothermal crystallization kinetics of cocoa butter. Thermochimica Acta

[22] Polish Standard. Confectionery products. Organoleptic evaluation. PN-A-88032 1998;

[23] Loisel C, Kelle G, Lecq G, Launay B, Ollivon M. Tempering of chocolate in a scraped

[24] Maat J, Rossi D, Babuchowski A, Beekmans F, Castenmiller J, Fenwick R, Haber J, Hogg T, Israelachwili D, Kettlitz B, Kohnke J, Lienemann K, Majou D, Petersen B, Schiefer G, Tomás-Barberán F. European Technology Platform on Food for Life. The Vision for 2020 and Beyond. Available; 2007 http://etp.ciaa.be/documents/SRA\_2007\_2010.pdf *(accessed*

[25] Budryn G, Nebesny E, Żyżelewicz D, Krysiak W, Motyl I, Libudzisz Z. Confectionery product of sugar-fat cores. Polish patent application: P-384154; 2007 (in polish). [26] Saarela M, Mogensen G, Fondén R, Mättö J, Mattila-Sandholm T. Probiotic bacteria: Safety, functional and technological properties. Journal of Biotechnology 2000;84 197-

[27] Besselich N. Die Keks-, Biskuit- und Waffelfabrikation. Verlag der Konditor-Zeitung,

[28] Manley D. Technology of Biscuits, Crackers and Cookies. Cambridge: Woodhead

[29] Warsza H. Wafer Products Manufacturing. Warsaw: Science-Technical Publications;

[30] Wyczański S. Confectionery Technology. Part 3. Warsaw: Ligot and Food Publications;

Food Sciences 2010;28(5) 392-406.

777/P05/2004/26; 2007 (in polish).

chocolate. LWT 2006;39 1053-1058.

of Texture Studies 1991; 22 219-229.

2003;400 131-142.

(in polish).

*11 June 2012).*

Trewir 1950;177-184, 188-190.

1965 pp. 177-179 (in polish).

215.

chocolates. International Chocolate Review 1958;13 290.

surface heat exchanger. Journal of Food Science 1997;62 773-780.

Publishing Limited, Boca Raton, CRC Press; 2000 pp. 430-436.

1970 pp. 5-7, 71-87, 93, 97, 101, 121-122 (in polish).

#### **8. References**

[15] Żyżelewicz D, Nebesny E, Motyl I, Libudzisz Z. Effect of milk chocolate supplementation with lyophilized *Lactobacillus* cells on its attributes. Czech Journal of Food Sciences 2010;28(5) 392-406.

304 Probiotics

**8. References**

*11 June 2012).*

of Intestinal Microbiology 2000;1 13-24.

report No R12 018 01; 2008 (in polish).

application: P-384866; 2008 (in polish).

application: P-388158; 2009 (in polish).

Expert Review of Anti-infective Therapy 2006;4: 261-275.

Gastroenterology 2003;17 849-859.

Metabolism 2007;21: 85-88.

We also would like to acknowledge help received from companies: ZPT "Kruszwica" S.A. (Kruszwica, Poland) and AARHUSKARLSHAMN SWEDEN AB (Karlshamn, Sweden) for

[1] FAO/WHO Report. Health and nutritional properties of probiotics in food including milk with live lactic acid bacteria. Report of a Joint FAO/WHO Expert Consultation.

[2] FAO/WHO Report. Guidelines for the Evaluation of Probiotics in Food. Report a Joint FAO/WHO Working Group. 2002. ftp://ftp.fao.org/es/esn/food/wgreport2.pdf *(accessed*

[3] Burns AJ, Rowland IR. Anti–carcinogenicity of probiotics and prebiotics. Current Issues

[4] Rafter J. Probiotics and colon cancer. Best Practice and Research Clinical

[5] Doron S, Gorbach SL. Probiotics: Their role in the treatment and prevention of disease.

[6] Boutron-Ruault MC. Probiotics and colorectal cancer. Clinical Nutrition and

[7] Nowak A, Libudzisz Z. Ability of intestinal lactic acid bacteria to bind or/and

[8] Nowak A, Arabski M, Libudzisz Z. Ability of intestinal lactic acid bacteria to bind or/and metabolise indole. Food Technology and Biotechnology 2008;46(3) 299-304. [9] Dicks LMT, Botes M. Probiotic lactic acid bacteria in the gastro-intestinal tract: Health

[10] Nebesny E, Żyżelewicz D, Krysiak W, Libudzisz Z, Motyl I. Physico-chemical, Microbiological and Organoleptic Properties of Sugar Free Chocolates Enriched with

[12] Krysiak W, Nebesny E, Żyżelewicz D, Budryn G, Motyl I, Libudzisz Z. Method of obtaining of pralines with sugar-fat filling of improved healthy properties. Polish patent

[13] Żyżelewicz D, Nebesny E, Krysiak W, Budryn G, Motyl I, Libudzisz Z. Spreadable product for bread of probiotic properties and preparation thereof. Polish patent

[14] Nebesny E, Żyżelewicz D, Motyl I, Libudzisz Z. Chocolate and preparation thereof. Polish Patent: P- 366273; 2012 (Patent Application, 2004) (in polish) – in press.

Viable Lactic Acid Bacteria. Grant's report No 3 P06T 054 24; 2005 (in polish). [11] Nebesny E, Żyżelewicz D, Budryn G, Krysiak W, Libudzisz Z, Motyl I. Confectionery Semi-products and Products Suplemented with Viable Lactic Acid Bacteria. Grant's

metabolize phenol and p-cresol. Annals of Microbiology 2007;57(3) 329-335.

Benefits, Safety and Mode of Action. Beneficial microbes 2010;1 11-29.

transferring a portion of fats used for obtaining nutty fatty mass and wafer creams.

2001. *www.who.int/foodsafety/.../fs.../probiotics.pdf (accessed 11 June 2012).*

[31] Żyżelewicz D, Nebesny E, Motyl I, Libudzisz Z, Budryn G, Krysiak W, Rosicka-Kaczmarek J. Confectionery product with functional properties. Wafers coated and noncoated with chocolate couverture, interleaved with filling supplemented with lactic acid bacteria (LAB). Patent application: P-393270; 2010 (in polish).

**Section 2**

**Probiotics in Health**

[32] Nebesny E, Żyżelewicz D, Krysiak W, Budryn G, Motyl I, Libudzisz Z. Raisins in chocolate coating. Polish patent application: P-384153; 2007 (in polish).

**Probiotics in Health**

306 Probiotics

[31] Żyżelewicz D, Nebesny E, Motyl I, Libudzisz Z, Budryn G, Krysiak W, Rosicka-Kaczmarek J. Confectionery product with functional properties. Wafers coated and noncoated with chocolate couverture, interleaved with filling supplemented with lactic acid

[32] Nebesny E, Żyżelewicz D, Krysiak W, Budryn G, Motyl I, Libudzisz Z. Raisins in

bacteria (LAB). Patent application: P-393270; 2010 (in polish).

chocolate coating. Polish patent application: P-384153; 2007 (in polish).

**Chapter 13**

© 2012 Mavroudi, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Mavroudi, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Probiotics in Pediatrics – Properties,**

Additional information is available at the end of the chapter

action and the indications for human use and health benefits.

Antigoni Mavroudi

http://dx.doi.org/10.5772/50043

**1. Introduction**

**Mechanisms of Action, and Indications**

Probiotics have been the topic of many studies over the past 20 years. Metchnikoff and Tissier (Metchnikoff 1907, Tissier, 1906) were the first to make scientific suggestions concerning the probiotic use of bacteria. They suggested that these bacteria could be administered to patients with diarrhea to help restore a healthy gut flora. Fuller (1989) in order to point out the microbial nature of probiotics redefined the word as "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance. " The most recent but probably not the last definition is "live microorganisms, which when consumed in adequate amounts, confer a health effect on the host"( Guarner and Schaafsma,1998). In the last 20 years however, research in the probiotic area has progressed considerably and significant advances have been made in selection and characterization of specific probiotic cultures. Most of the studies aim to investigate the physiological and functional properties of various probiotic strains, the mechanisms of

Probiotic bacteria are a subset of specific organisms, which, when ingested, transiently occupy the gastrointestinal tract and lead to documented health benefits. Lactic-acidproducing bacteria (LAB), particularly members of the genus Lactobacilli, Bifidobacteria, non pathogenic gram positive bacteria and non bacterial microorganisms (for example certain yeasts, such as Saccharomyces boulardii) have been used as probiotic agents. [1] The use of specific probiotic bacteria has been shown to enhance host defense mechanisms. [2] Prebiotics are non-digestable food ingredients that beneficially affect the host by stimulating the growth and/or activity of a limited number of bacterial species in the colon. Compounds most commonly studied for their prebiotic nature are non-digestable carbohydrates. In particular, oligosaccharides are considered the main units among prebiotics, which include fructooligosaccharides (FOS), inulin, lactulose and galactooligosaccharides (GOS). Synbiotics

## **Probiotics in Pediatrics – Properties, Mechanisms of Action, and Indications**

Antigoni Mavroudi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50043

## **1. Introduction**

Probiotics have been the topic of many studies over the past 20 years. Metchnikoff and Tissier (Metchnikoff 1907, Tissier, 1906) were the first to make scientific suggestions concerning the probiotic use of bacteria. They suggested that these bacteria could be administered to patients with diarrhea to help restore a healthy gut flora. Fuller (1989) in order to point out the microbial nature of probiotics redefined the word as "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance. " The most recent but probably not the last definition is "live microorganisms, which when consumed in adequate amounts, confer a health effect on the host"( Guarner and Schaafsma,1998). In the last 20 years however, research in the probiotic area has progressed considerably and significant advances have been made in selection and characterization of specific probiotic cultures. Most of the studies aim to investigate the physiological and functional properties of various probiotic strains, the mechanisms of action and the indications for human use and health benefits.

Probiotic bacteria are a subset of specific organisms, which, when ingested, transiently occupy the gastrointestinal tract and lead to documented health benefits. Lactic-acidproducing bacteria (LAB), particularly members of the genus Lactobacilli, Bifidobacteria, non pathogenic gram positive bacteria and non bacterial microorganisms (for example certain yeasts, such as Saccharomyces boulardii) have been used as probiotic agents. [1] The use of specific probiotic bacteria has been shown to enhance host defense mechanisms. [2] Prebiotics are non-digestable food ingredients that beneficially affect the host by stimulating the growth and/or activity of a limited number of bacterial species in the colon. Compounds most commonly studied for their prebiotic nature are non-digestable carbohydrates. In particular, oligosaccharides are considered the main units among prebiotics, which include fructooligosaccharides (FOS), inulin, lactulose and galactooligosaccharides (GOS). Synbiotics

© 2012 Mavroudi, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Mavroudi, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

are a combination of probiotics and prebiotics and it is the synergy between these two substances that becomes known as synbiotics.

Probiotics in Pediatrics – Properties, Mechanisms of Action, and Indications 311

The intestine of the newborn is essentially sterile. During the birthing process and during the first days of life, the gut is inoculated with bacteria. In the first two days of life, an infant's intestinal tract is rapidly colonized with bacteria consisting mainly of Enterobacteria. In most breastfed infants, the Bifidobacteria counts increase rapidly to constitute 80-90% of the total flora. Formula-fed infants, on the other hand, tent to have a flora that is more complex, consisting mostly of coliforms and Bacteroides with significantly lower prevalence of Bifidobacteria. [7] Although the composition of the microflora varies among individuals, the composition within each individual remains stable over prolonged periods. [8] A normal microbial flora is necessary for the development of gut associated lymphoid tissue (GALT). The gut luminal microbes are responsible for mucosal immune system development in healthy infants. Signaling through specific receptors, particularly toll-like receptors, intestinal bacteria affect epithelium cell function, which determines T-cell differentiation and antibody responses to T-cell-dependent antigens, regulating immune gut response for IgA responses to luminal antigens. [9] Resident bacteria, particularly Lactobacilli and Bifidobacteria, can exert antimicrobial activities influencing both local and

Intestinal bacteria have a major effect on enhancing secretory immune function. Among the more consistently found effects of specific Bifidobacteria and Lactobacilli in pediatric populations is the effect on humoral immunity, particularly on secretory IgA( s IgA ) and other immunoglobulins. An increase in IgA-, IgM-,and IgG-secreting cells in circulation ,as well as fecal IgA concentrations ,has been reported. During the neonatal period, s IgA in the stool of formula-fed infants is essentially undetectable. [11, 12] Bifidobacteria and Lactobacillus given orally have been shown to influence s IgA in a number of animal trials [13] Infant studies that investigated the effects of specific Lactobacilli and Bifidobacteria supplementation on stimulating the mucosal immune response have reported similar positive results. Breast milk contains significant levels of sIgA that are transferred to the infant. Bifidobacteria, which predominate in breast-fed infants, have shown to stimulate the synthesis and secretion of IgA. Recent reports indicate similar IgA increases in premature infants receiving B lactis. [14] sIgA, the most important and predominant immunoglobulin in mucosal surfaces, provides protection against antigens, potential pathogens, toxins, and

The resident Bifidobacteria and Lactobacilli in the gut can offer resistance to colonization by other potentially pathogenic microbes, thereby functioning as part of the gut defense barrier. They have also been associated with the secretion of substrates that have antimicrobial properties [16] and the secretion of mucins via activation of MUC2 and MUC3 genes, part of

An increasing number of clinical trials have documented effects of ingestion of specific probiotic bacteria on gut barrier function and immunity. For example in both animal and human models, ingestion of L casei, L bulgaricus, and L acidophilus has been shown to activate production of macrophages and enhance phagocytosis. [8] Serum sCD14, a marker

the intestinal barrier that can inhibit adherence of pathogenic bacteria. [17]

**3. Mechanisms of action**

systemic immunity. [10]

virulence factors. [15]

Several clinical benefits have been reported as a result of interaction between host and becteria ,such as for treatment and prevention of viral diarrhea [3] and reducing the risk of necrotizing enterocolitis (NEC), mitigating antibiotic associated diarrhea ,and modulating host immune response (such as in allergic disease ).

## **2. Properties**

Intestinal microflora is composed of both well-established resident microbes and those ingested orally which transiently occupy the gastrointestinal (GI) tract. Probiotics are generally defined as non pathogenic organisms in food supply (ingested microbes) that are capable of conferring a health benefit to the host by modifying gut microbial ecology.

Probiotics are live microorganisms which when ingested in adequate amounts confer a health effect on the host by enhancing host defense mechanisms. Several clinical benefits have been reported with various specific microbes in pediatric populations. It is increasingly clear that these benefits to the host are mostly mediated by the profound effect that intestinal microflora (microbiota) have on gut barrier function and host immune response. The most frequently used probiotic agents are the lactic acid producing bacteria, such as Lactobacilli and Bifidobacteria, non pathogenic strains of Gram positive bacteria, such as Streptococcus, E. Coli and non bacterial microorganisms, such as Saccharomyces Bulardii

There are several generally accepted characteristics that define probiotic bacteria. [4-6]

Probiotics can be found in certain foods, such as yogurts, fruit juices and soy beverages. They are also found in supplements that come in liquid, capsule and powdered forms. It is believed that a concentration of 10 live microorganisms per gram or ml of product is required in order to exert a health benefit on the host.

Probiotics have a wide range of beneficial effects and numerous indications of use in pediatric populations, such as:

### **3. Mechanisms of action**

310 Probiotics

**2. Properties**

They are microbial organisms

pediatric populations, such as:

Antibiotic-Associated Diarrhea

Acute diarrhea

 Allergy prevention Necrotizing enterocolitis

They survive gastric, biliary, and pancreatic digestion.

should "colonize" the intestine. [5,6]

required in order to exert a health benefit on the host.

functional and clinical benefit to the host when consumed.

substances that becomes known as synbiotics.

host immune response (such as in allergic disease ).

are a combination of probiotics and prebiotics and it is the synergy between these two

Several clinical benefits have been reported as a result of interaction between host and becteria ,such as for treatment and prevention of viral diarrhea [3] and reducing the risk of necrotizing enterocolitis (NEC), mitigating antibiotic associated diarrhea ,and modulating

Intestinal microflora is composed of both well-established resident microbes and those ingested orally which transiently occupy the gastrointestinal (GI) tract. Probiotics are generally defined as non pathogenic organisms in food supply (ingested microbes) that are

Probiotics are live microorganisms which when ingested in adequate amounts confer a health effect on the host by enhancing host defense mechanisms. Several clinical benefits have been reported with various specific microbes in pediatric populations. It is increasingly clear that these benefits to the host are mostly mediated by the profound effect that intestinal microflora (microbiota) have on gut barrier function and host immune response. The most frequently used probiotic agents are the lactic acid producing bacteria, such as Lactobacilli and Bifidobacteria, non pathogenic strains of Gram positive bacteria, such as Streptococcus, E. Coli and non bacterial microorganisms, such as Saccharomyces Bulardii

capable of conferring a health benefit to the host by modifying gut microbial ecology.

There are several generally accepted characteristics that define probiotic bacteria. [4-6]

They remain viable and stable after culture manipulation, and storage before consumption

 They are able to induce a host response once they enter the intestinal microbial ecosystem (by adhering to gut epithelium or other mechanisms) and they yield a

It has been suggested that probiotic bacteria should be of "human origin" and that they

Probiotics can be found in certain foods, such as yogurts, fruit juices and soy beverages. They are also found in supplements that come in liquid, capsule and powdered forms. It is believed that a concentration of 10 live microorganisms per gram or ml of product is

Probiotics have a wide range of beneficial effects and numerous indications of use in

The intestine of the newborn is essentially sterile. During the birthing process and during the first days of life, the gut is inoculated with bacteria. In the first two days of life, an infant's intestinal tract is rapidly colonized with bacteria consisting mainly of Enterobacteria. In most breastfed infants, the Bifidobacteria counts increase rapidly to constitute 80-90% of the total flora. Formula-fed infants, on the other hand, tent to have a flora that is more complex, consisting mostly of coliforms and Bacteroides with significantly lower prevalence of Bifidobacteria. [7] Although the composition of the microflora varies among individuals, the composition within each individual remains stable over prolonged periods. [8] A normal microbial flora is necessary for the development of gut associated lymphoid tissue (GALT). The gut luminal microbes are responsible for mucosal immune system development in healthy infants. Signaling through specific receptors, particularly toll-like receptors, intestinal bacteria affect epithelium cell function, which determines T-cell differentiation and antibody responses to T-cell-dependent antigens, regulating immune gut response for IgA responses to luminal antigens. [9] Resident bacteria, particularly Lactobacilli and Bifidobacteria, can exert antimicrobial activities influencing both local and systemic immunity. [10]

Intestinal bacteria have a major effect on enhancing secretory immune function. Among the more consistently found effects of specific Bifidobacteria and Lactobacilli in pediatric populations is the effect on humoral immunity, particularly on secretory IgA( s IgA ) and other immunoglobulins. An increase in IgA-, IgM-,and IgG-secreting cells in circulation ,as well as fecal IgA concentrations ,has been reported. During the neonatal period, s IgA in the stool of formula-fed infants is essentially undetectable. [11, 12] Bifidobacteria and Lactobacillus given orally have been shown to influence s IgA in a number of animal trials [13] Infant studies that investigated the effects of specific Lactobacilli and Bifidobacteria supplementation on stimulating the mucosal immune response have reported similar positive results. Breast milk contains significant levels of sIgA that are transferred to the infant. Bifidobacteria, which predominate in breast-fed infants, have shown to stimulate the synthesis and secretion of IgA. Recent reports indicate similar IgA increases in premature infants receiving B lactis. [14] sIgA, the most important and predominant immunoglobulin in mucosal surfaces, provides protection against antigens, potential pathogens, toxins, and virulence factors. [15]

The resident Bifidobacteria and Lactobacilli in the gut can offer resistance to colonization by other potentially pathogenic microbes, thereby functioning as part of the gut defense barrier. They have also been associated with the secretion of substrates that have antimicrobial properties [16] and the secretion of mucins via activation of MUC2 and MUC3 genes, part of the intestinal barrier that can inhibit adherence of pathogenic bacteria. [17]

An increasing number of clinical trials have documented effects of ingestion of specific probiotic bacteria on gut barrier function and immunity. For example in both animal and human models, ingestion of L casei, L bulgaricus, and L acidophilus has been shown to activate production of macrophages and enhance phagocytosis. [8] Serum sCD14, a marker

of immunologic maturation in the neonate, was significantly greater than placebo in infants provided probiotics. Additionally, decreased gut permeability with Lactobacilli [18] , and recently in premature infants receiving Bifidobacteria [19] , is another mechanism by which probiotics may function.

Probiotics in Pediatrics – Properties, Mechanisms of Action, and Indications 313

In summary effects have been documented supported by a large body of evidence from in vitro and animal studies. These include effects on innate (nonspecific immune defense) and adaptive immunity (responses that require exposure to pathogens or antigens that the immune system recognizes and "remembers"). Adequate adaptive responses are important for host defense, as well as to develop immune tolerance, which decreases chances for abnormal immune hyperreactivity or inflammation. The following effects on innate and

Enhance natural killer cell activity, macrophage stimulation, and phagocytosis

Clinical benefits with specific probiotic bacteria by enhancing defense mechanisms, as well as by modulating host immune response include prevention and treatment of acute infectious diarrhea and antibiotic-associated diarrhea, modulating allergic immune

The clinical outcome with the use of probiotic bacteria in order to treat or prevent acute diarrheal diseases has been supported by a large and growing body of evidence. The larger number of trials documents therapeutic use of probiotics as supplements early in the course of the disease. The rationale of using probiotics to treat and prevent diarrheal diseases is based on the assumption that they modify the composition of colonic microflora and act against enteric pathogens. The majority of studies have included various species of Lactobacilli, and by far the most used has been L rhamnosus (GG). This specific strain has shown efficacy when given as a supplement early in the course of rotaviral diarrhea. The most consistent effect reported is a reduction in duration of illness (0, 5 to 1, 5 days). While for the individual infant the effect may be modest, the effect on the population may be

A reduction in incidence of acute diarrheal disease has been reported by another body of literature. Several studies have documented a reduction in incidence or severity of acute diarrhea with Bifidobacteria mainly B. lactis [39, 40] and with Lactobacilli, mainly L rhamnosus (GG) [41, 42] though protection is not always significant. [43] Both L rhamnosus

adaptive immunity have been reported:

Compete with and inhibit growth of potential pathogens

Increase total and specific s IgA in serum and intestinal lumen

Increase IgA-, IgG-, and IgM- secreting cells

Modulate inflammatory gut immune responses [5]

response, prevention of NEC and treating constipation.

*Effects on innate immunity*

 Promote mucin production Decrease gut permeability

**4.1. Acute infectious diarrhea**

*Effects on adaptive immunity*

**4. Indications**

significant. [38]

Some probiotic bacteria have been shown to exert beneficial effects on pro- and antiinflammatory cytokine secretion [8]. Decreases in fecal 1 antitrypsin, urinary protein eosinophil X, tumor necrosis factor (TNF)-α [20,21] have been reported as a result of downregulation of the inflammatory immune response by probiotic agents.

It is being recognized that host-microbe interactions have an effect on atopic disease. Alterations in the balance of intestinal microflora, particularly in immune and inflammatory-related diseases coupled with significant reduction in the oral ingestion and exposure to a microbe that has led to postulation of the "hygiene hypothesis". This theory suggests that a lower exposure in early childhood to bacterial and other antigens in industrialized societies has led to inadequate development and maturation of immune responses and appears responsible for the increased prevalence of asthma and allergies due to inadequate defensive and immune-modulating gut immune diseases. [22, 23, 24] Infants are born with a predominance of Th2 (T helper 2) lymphocyte activity ,which predisposes them to an exaggerated response to allergens ,with increased IgE production. Exposure to intestinal bacteria ,on the other hand ,stimulates Th1 ( T helper 1 ) activity ( which primarily reacts defensively to bacterial stimuli as part of the protective immune response ). As a consequence ,intestinal microbes ( resident and ingested )can redirect immune balance from a Th2-predominant response to a balanced Th1/Th2 response ,decreasing the changes for a potential exaggerated allergic response. Finally, TReg (regulatory) cells release cytokines such as transforming growth factor β(TGF-β) ,which can inhibit Th1 or Th2 overexpression and also play a role in adequate balancing the host response to bacterial food antigens ,and their activity seems to be increased by luminal microbes [25,26,27,28] Some Bifidobacteria and Lactobacilli given orally may enhance the production of a balanced T-helper-cell response [29,30] and stimulate production of interleukin (IL)-10, and TGF-β [21,31,32] both of which have a role in the development of immunologic tolerance to antigens and can decrease allergic type immune responses.

Bifidobacteria supplementation in premature infants has been shown to positively modify the microflora of the intestines. [33] Beneficial increases in stool, short-chain fatty acids, reductions in stool pH, and decreases in fecal ammonia and indoles [34, 35] and concentrations of Bacteroides and E. Coli have been documented [36, 37] with Bifidobacteria supplementation. Specific probiotic bacteria positively affect the ratio of favorable to unfavorable in the gut luminal environment. Lactobacilli and Bifidobacteria when ingested they are not part of the resident microflora of the host, and their counts typically decrease or disappear once ingestion stops. Specific Lactobacilli and Bifidobacteria, when ingested, can modify the composition of intestinal microbial ecology. They are not typically pathogenic and seem beneficial in fostering host immune development and response. These ingested organisms have the potential of further supporting gut barrier function and appropriate host immune system development and immune response.

In summary effects have been documented supported by a large body of evidence from in vitro and animal studies. These include effects on innate (nonspecific immune defense) and adaptive immunity (responses that require exposure to pathogens or antigens that the immune system recognizes and "remembers"). Adequate adaptive responses are important for host defense, as well as to develop immune tolerance, which decreases chances for abnormal immune hyperreactivity or inflammation. The following effects on innate and adaptive immunity have been reported:

#### *Effects on innate immunity*

312 Probiotics

probiotics may function.

decrease allergic type immune responses.

host immune system development and immune response.

of immunologic maturation in the neonate, was significantly greater than placebo in infants provided probiotics. Additionally, decreased gut permeability with Lactobacilli [18] , and recently in premature infants receiving Bifidobacteria [19] , is another mechanism by which

Some probiotic bacteria have been shown to exert beneficial effects on pro- and antiinflammatory cytokine secretion [8]. Decreases in fecal 1 antitrypsin, urinary protein eosinophil X, tumor necrosis factor (TNF)-α [20,21] have been reported as a result of down-

It is being recognized that host-microbe interactions have an effect on atopic disease. Alterations in the balance of intestinal microflora, particularly in immune and inflammatory-related diseases coupled with significant reduction in the oral ingestion and exposure to a microbe that has led to postulation of the "hygiene hypothesis". This theory suggests that a lower exposure in early childhood to bacterial and other antigens in industrialized societies has led to inadequate development and maturation of immune responses and appears responsible for the increased prevalence of asthma and allergies due to inadequate defensive and immune-modulating gut immune diseases. [22, 23, 24] Infants are born with a predominance of Th2 (T helper 2) lymphocyte activity ,which predisposes them to an exaggerated response to allergens ,with increased IgE production. Exposure to intestinal bacteria ,on the other hand ,stimulates Th1 ( T helper 1 ) activity ( which primarily reacts defensively to bacterial stimuli as part of the protective immune response ). As a consequence ,intestinal microbes ( resident and ingested )can redirect immune balance from a Th2-predominant response to a balanced Th1/Th2 response ,decreasing the changes for a potential exaggerated allergic response. Finally, TReg (regulatory) cells release cytokines such as transforming growth factor β(TGF-β) ,which can inhibit Th1 or Th2 overexpression and also play a role in adequate balancing the host response to bacterial food antigens ,and their activity seems to be increased by luminal microbes [25,26,27,28] Some Bifidobacteria and Lactobacilli given orally may enhance the production of a balanced T-helper-cell response [29,30] and stimulate production of interleukin (IL)-10, and TGF-β [21,31,32] both of which have a role in the development of immunologic tolerance to antigens and can

Bifidobacteria supplementation in premature infants has been shown to positively modify the microflora of the intestines. [33] Beneficial increases in stool, short-chain fatty acids, reductions in stool pH, and decreases in fecal ammonia and indoles [34, 35] and concentrations of Bacteroides and E. Coli have been documented [36, 37] with Bifidobacteria supplementation. Specific probiotic bacteria positively affect the ratio of favorable to unfavorable in the gut luminal environment. Lactobacilli and Bifidobacteria when ingested they are not part of the resident microflora of the host, and their counts typically decrease or disappear once ingestion stops. Specific Lactobacilli and Bifidobacteria, when ingested, can modify the composition of intestinal microbial ecology. They are not typically pathogenic and seem beneficial in fostering host immune development and response. These ingested organisms have the potential of further supporting gut barrier function and appropriate

regulation of the inflammatory immune response by probiotic agents.

#### *Effects on adaptive immunity*

#### **4. Indications**

Clinical benefits with specific probiotic bacteria by enhancing defense mechanisms, as well as by modulating host immune response include prevention and treatment of acute infectious diarrhea and antibiotic-associated diarrhea, modulating allergic immune response, prevention of NEC and treating constipation.

#### **4.1. Acute infectious diarrhea**

The clinical outcome with the use of probiotic bacteria in order to treat or prevent acute diarrheal diseases has been supported by a large and growing body of evidence. The larger number of trials documents therapeutic use of probiotics as supplements early in the course of the disease. The rationale of using probiotics to treat and prevent diarrheal diseases is based on the assumption that they modify the composition of colonic microflora and act against enteric pathogens. The majority of studies have included various species of Lactobacilli, and by far the most used has been L rhamnosus (GG). This specific strain has shown efficacy when given as a supplement early in the course of rotaviral diarrhea. The most consistent effect reported is a reduction in duration of illness (0, 5 to 1, 5 days). While for the individual infant the effect may be modest, the effect on the population may be significant. [38]

A reduction in incidence of acute diarrheal disease has been reported by another body of literature. Several studies have documented a reduction in incidence or severity of acute diarrhea with Bifidobacteria mainly B. lactis [39, 40] and with Lactobacilli, mainly L rhamnosus (GG) [41, 42] though protection is not always significant. [43] Both L rhamnosus

(GG) and L reuteri (during treatment) [44] and B lactis (used prophylactically) [45] have documented reduced rotaviral shedding. Thirty-four randomized clinical trials reviewed by a meta-analysis evaluated the efficacy of probiotics in the prevention of acute diarrhea. Probiotics significantly reduced the risk of diarrhea developing in infants and children by 57%. The protective effect did not significantly vary among the probiotic strains used, including B lactis, L rhamnosus GG, L acidophilus, S bouladrii, and other agents used alone or in combination with 2 or more strains. [46] Decreased hospitalization [47] and reduced duration of hospitalization were also confirmed. All studies suggested that the effect occurs on both the manifestations of the disease and on the course of the infection. There has been no study so far documenting an increase in diarrheal disease with probiotic use. These findings suggest that specific probiotics may be used in a long-term and prophylactic manner, particularly in infancy.

Probiotics in Pediatrics – Properties, Mechanisms of Action, and Indications 315

indicated that concomitant treatment with probiotics, compared with placebo reduced the risk of diarrhea from 28, 5% to 11, 9%. [67] Beneficial effects were strongest for B lactis and S

In conclusion, Randomized Controlled Trials (RCTs) in children have provided so far evidence of a moderate beneficial effect of L rhamnosus (GG), B. lactis, S. thermophilus and S. boulardii in preventing AAD. No data on efficacy of other probiotic strains are available in children. Based on the previously reported evidence probiotics have been shown capable of providing reasonable protection against the development of AAD. Their use is probably warranted whenever the physician feels that preventing this usually self-limited

Nosocomial diarrhea refers to any diarrhea contracted in a health care institution and is more commonly caused by enteric pathogens especially rotavirus. [68] The reported incidence ranges from 4, 5 to 22, 6 episodes per 100 admissions. It may prolong hospital stays and increase medical costs. Although hand washing is the essential infection control measure, other cost-effective approaches to prevent nosocomial diarrhea are being

Two RCTs evaluated the use of L rhamnosus G [69, 70] on nosocomial diarrhea prevention. The first study showed that L rhamnosus G administered orally twice daily significantly reduced the risk of diarrhea compared with placebo (6, 7% vs 33, 3%; p=0,002) [69]. The second RCT evaluating L rhamnosus G in the prevention of diarrhea involved 220 children. L rhamnosus (GG) was administered orally once daily and did not prevent nosocomial rotavirus infections compared with placebo (25, 4% vs 30, 2%; p=0, 4). However, the rate of symptomatic rotavirus enteritis was lower in children receiving L rhamnosus (GG)

The available data do not provide strong evidence for the routine use of L rhamnosus (GG)

Two other RCTs evaluated the efficacy of B. bifidum and S. thermophilus in preventing nosocomial diarrhea. The first study showed that the administration of standard infant formula supplemented with B. bifidum and S. thermophilus reduced the prevalence of nosocomial diarrhea compared with placebo. The risk of rotavirus gastroenteritis was significantly lower in those receiving probiotic-supplemented formula [71]. The second RCT showed that infants living in residential care settings, although they differ from hospital settings are also at increased risk for diarrheal illnesses, and the mode of acquiring diarrhea is similar. The infants received milk formula supplemented with viable B. lactis strain Bb12. It was shown that the previous intervention did not reduce the prevalence of diarrhea

In conclusion there is conflicting evidence on the efficacy of L rhamnosus (GG) provided by 2 RCTs in preventing nosocomial diarrhea. One small RCT suggests a benefit of B. bifidum

thermophilus given in infant formula and L rhamnosus (GG) as a supplement.

complication is important.

**4.3. Nosocomial diarrhea**

compared to placebo. [72]

compared with placebo (13% vs 21%; p=0, 13). [70]

to prevent nosocomial rotavirus diarrhea in infants and toddlers.

evaluated.

Several mechanisms have been proposed in order to explain the efficacy of probiotics in preventing or treating acute diarrhea. It has been shown that probiotics stimulate or modify nonspecific and specific immune responses to pathogens. Probiotics have been shown to enhance mucosal immune defenses and protect structural and functional damage promoted by enteric pathogens in the brush border of enterocytes, probably by interfering with the cross-talk between the pathogen and host cells. [48] It has been shown that L rhamnosus (GG) and Lactobacillus plantarum 299v inhibit, in a dose-dependent manner, the binding of E.coli to intestinal-derived epithelial cells grown in tissue culture by stimulation of synthesis and secretion of mucins. [49] Certain probiotics increase the number of circulating lymphocytes [50] and lymphocyte proliferation [51,] stimulate phagocytosis, increase specific antibody responses to rotavirus vaccine strain [52] , and increase cytokine secretion, including interferon-γ. [51] L rhamnosus GG and Lactobacillus acidophilus have been shown to produce antimicrobial substances against some gram-positive and gram-negative pathogens. [53, 54] Other mechanisms proposed by which probiotics might exert their activity against pathogens are competition for nutrients required for growth of pathogens [55,56] ,competitive inhibition of adhesion of pathogens [57-60] ,and modification of toxins and toxin receptors. [61,62]

#### **4.2. Antibiotic-associated diarrhea**

Antibiotic-associated diarrhea (AAD) is defined as an acute inflammation of the intestinal mucosa caused by the administration of a broad spectrum of antibiotics. The single bacterial agent most commonly associated with AAD is Clostiridium difficile. However, when the normal fecal gram-negative organisms are absent, overgrowth by staphylococci, yeasts and fungi has been implicated. [63] In fact, most episodes of AAD in childhood are not due to C. difficile. [64] The rationale for the use of probiotics in AAD is based on the assumption that the key factor in the pathogenesis of AAD is a disturbance in normal intestinal flora.

Several probiotic bacteria have proved to be beneficial in reducing the risk of antibioticassociated diarrhea in infants and children. [65-67] Six randomized controlled trials that collectively assessed 766 children for the efficacy of probiotics in the prevention of AAD indicated that concomitant treatment with probiotics, compared with placebo reduced the risk of diarrhea from 28, 5% to 11, 9%. [67] Beneficial effects were strongest for B lactis and S thermophilus given in infant formula and L rhamnosus (GG) as a supplement.

In conclusion, Randomized Controlled Trials (RCTs) in children have provided so far evidence of a moderate beneficial effect of L rhamnosus (GG), B. lactis, S. thermophilus and S. boulardii in preventing AAD. No data on efficacy of other probiotic strains are available in children. Based on the previously reported evidence probiotics have been shown capable of providing reasonable protection against the development of AAD. Their use is probably warranted whenever the physician feels that preventing this usually self-limited complication is important.

#### **4.3. Nosocomial diarrhea**

314 Probiotics

manner, particularly in infancy.

and toxin receptors. [61,62]

**4.2. Antibiotic-associated diarrhea**

(GG) and L reuteri (during treatment) [44] and B lactis (used prophylactically) [45] have documented reduced rotaviral shedding. Thirty-four randomized clinical trials reviewed by a meta-analysis evaluated the efficacy of probiotics in the prevention of acute diarrhea. Probiotics significantly reduced the risk of diarrhea developing in infants and children by 57%. The protective effect did not significantly vary among the probiotic strains used, including B lactis, L rhamnosus GG, L acidophilus, S bouladrii, and other agents used alone or in combination with 2 or more strains. [46] Decreased hospitalization [47] and reduced duration of hospitalization were also confirmed. All studies suggested that the effect occurs on both the manifestations of the disease and on the course of the infection. There has been no study so far documenting an increase in diarrheal disease with probiotic use. These findings suggest that specific probiotics may be used in a long-term and prophylactic

Several mechanisms have been proposed in order to explain the efficacy of probiotics in preventing or treating acute diarrhea. It has been shown that probiotics stimulate or modify nonspecific and specific immune responses to pathogens. Probiotics have been shown to enhance mucosal immune defenses and protect structural and functional damage promoted by enteric pathogens in the brush border of enterocytes, probably by interfering with the cross-talk between the pathogen and host cells. [48] It has been shown that L rhamnosus (GG) and Lactobacillus plantarum 299v inhibit, in a dose-dependent manner, the binding of E.coli to intestinal-derived epithelial cells grown in tissue culture by stimulation of synthesis and secretion of mucins. [49] Certain probiotics increase the number of circulating lymphocytes [50] and lymphocyte proliferation [51,] stimulate phagocytosis, increase specific antibody responses to rotavirus vaccine strain [52] , and increase cytokine secretion, including interferon-γ. [51] L rhamnosus GG and Lactobacillus acidophilus have been shown to produce antimicrobial substances against some gram-positive and gram-negative pathogens. [53, 54] Other mechanisms proposed by which probiotics might exert their activity against pathogens are competition for nutrients required for growth of pathogens [55,56] ,competitive inhibition of adhesion of pathogens [57-60] ,and modification of toxins

Antibiotic-associated diarrhea (AAD) is defined as an acute inflammation of the intestinal mucosa caused by the administration of a broad spectrum of antibiotics. The single bacterial agent most commonly associated with AAD is Clostiridium difficile. However, when the normal fecal gram-negative organisms are absent, overgrowth by staphylococci, yeasts and fungi has been implicated. [63] In fact, most episodes of AAD in childhood are not due to C. difficile. [64] The rationale for the use of probiotics in AAD is based on the assumption that

the key factor in the pathogenesis of AAD is a disturbance in normal intestinal flora.

Several probiotic bacteria have proved to be beneficial in reducing the risk of antibioticassociated diarrhea in infants and children. [65-67] Six randomized controlled trials that collectively assessed 766 children for the efficacy of probiotics in the prevention of AAD Nosocomial diarrhea refers to any diarrhea contracted in a health care institution and is more commonly caused by enteric pathogens especially rotavirus. [68] The reported incidence ranges from 4, 5 to 22, 6 episodes per 100 admissions. It may prolong hospital stays and increase medical costs. Although hand washing is the essential infection control measure, other cost-effective approaches to prevent nosocomial diarrhea are being evaluated.

Two RCTs evaluated the use of L rhamnosus G [69, 70] on nosocomial diarrhea prevention. The first study showed that L rhamnosus G administered orally twice daily significantly reduced the risk of diarrhea compared with placebo (6, 7% vs 33, 3%; p=0,002) [69]. The second RCT evaluating L rhamnosus G in the prevention of diarrhea involved 220 children. L rhamnosus (GG) was administered orally once daily and did not prevent nosocomial rotavirus infections compared with placebo (25, 4% vs 30, 2%; p=0, 4). However, the rate of symptomatic rotavirus enteritis was lower in children receiving L rhamnosus (GG) compared with placebo (13% vs 21%; p=0, 13). [70]

The available data do not provide strong evidence for the routine use of L rhamnosus (GG) to prevent nosocomial rotavirus diarrhea in infants and toddlers.

Two other RCTs evaluated the efficacy of B. bifidum and S. thermophilus in preventing nosocomial diarrhea. The first study showed that the administration of standard infant formula supplemented with B. bifidum and S. thermophilus reduced the prevalence of nosocomial diarrhea compared with placebo. The risk of rotavirus gastroenteritis was significantly lower in those receiving probiotic-supplemented formula [71]. The second RCT showed that infants living in residential care settings, although they differ from hospital settings are also at increased risk for diarrheal illnesses, and the mode of acquiring diarrhea is similar. The infants received milk formula supplemented with viable B. lactis strain Bb12. It was shown that the previous intervention did not reduce the prevalence of diarrhea compared to placebo. [72]

In conclusion there is conflicting evidence on the efficacy of L rhamnosus (GG) provided by 2 RCTs in preventing nosocomial diarrhea. One small RCT suggests a benefit of B. bifidum and S. thermophilus in sick infants admitted to the hospital, but no such benefit has been identified in healthy children in residential care settings. The already mentioned studies suggest that there is currently not enough evidence to recommend the routine use of probiotics to prevent nosocomial diarrhea. There is a need for large and well-designed RCTs.

Probiotics in Pediatrics – Properties, Mechanisms of Action, and Indications 317

effect against antigens that might otherwise ultimately lead to systemic allergic symptoms

Proliferation and growth of beneficial bacteria in the digestive system is being promoted with the use of prebiotics. Prebiotics are generally considered to be safe and they are naturally present in several kinds of food. A food ingredient must fulfill the following criteria to be considered a prebiotic: it should be hydrolyzed or absorbed in the upper part of the gastrointestinal tract, it has to be a selective substrate for beneficial bacteria in the colon for example bifidobacteria, and it must be able to alter the intestinal microflora

In regards to the immunomodulatory effect of prebiotics, the proposed mechanisms of action are the following: They are thought to stimulate the activity of lactic acid bacteria, such as lactobacilli and bifidobacteria, which have immunomodulatory qualities. A second mechanism of action is that fermentation of prebiotics by lactic acid bacteria enhances Short Chain Fatty Acids (SCFA) that they act as energy substrate for colonocytes. It has been

The immunomodulatory effect of prebiotics on the prevention of atopic dermatitis has been evaluated by several studies. A study by Moro et al showed that a mixture of prebiotic oligosaccharides reduces the incidence of atopic dermatitis during the first six months of age [85]. A study by van der Aa et al determined the effect of Bifidobacterium breve M-16V combined with a prebiotic oligosaccharide mixture (synbiotic) on atopic markers. The synbiotic mixture had no detectable effect on plasma levels of the analysed atopic disease markers in vivo [86]. Another study by de Kivit S, et al investigated the effect of prebiotic galacto- and fructo-oligosaccharides (scGOS/lcFOS) in combination with Bifidobacterium breve M-16V (GF/Bb) on atopy. The study showed that dietary supplementation with GF/Bb enhances serum galectin-9 levels, which associates with the prevention of the allergic

In conclusion, although theoretically pro-, pre and synbiotics are promising candidates to prevent or treat AD, results of the clinical trials performed so far are not conclusive. Prevention trials show promising but heterogenic results. Therefore at this moment there is not enough evidence to support the use of pro-, pre-, or synbiotics for prevention of AD in clinical practice. Results of treatment trials are not very convincing, however pro- or synbiotics could possibly play a role in the treatment of IgE-associated AD, which should be

Microflora establishment and composition in premature infants is a major determinant in the pathophysiology of NEC. The premature infant is exposed to a variety of factors that negatively affect their possibilities of attaining an appropriate colonization. These factors include increasing exposure to potential delayed colonization, colonization with "neonatal intensive care unit environmental microbes", use of antibiotics, lack of exposure to maternal

shown that SCFA stimulate Interferon-γ and IL-10 production. [81-84]

such as eczema. [37]

symptoms. [87]

elucidated in future prospective trials.

**4.5. Necrotizing enterocololitis**

flora and breast milk.

towards a healthier composition [80].

#### **4.4. Allergy**

The rationale for using probiotics in prevention and treatment of allergic disorders is based on the concept that appropriate microbial stimuli are required for normal early immunological development. Microbial-gut interactions can improve the integrity of the gut barrier by decreasing intestinal permeability, reducing both adherence of potential antigens and their systemic effect, and by modulating GALT immune response toward antigen tolerance. The intestinal microflora interacts with the mucosal immune system. It has been found that different strains of commercial bacteria vary in the cytokine response they generate. The Th1/Th2 imbalance is crucial to the clinical expression of allergy. Probiotic bacteria can produce significant antiallergenic effects by intricate interactions inducing Th1 cytokines, such as interferon-γ [73] , Τ-regulatory cytokines, such as IL-10 and TGF-β [74] , and mucosal immunoglobulin A production [75].

Three species of Lactobacillus were shown to modulate the phenotype and functions of human myeloid dendritic cells (DCs). Lactobacillus-exposed myeloid DCs up-regulated HLA-DR, CD83, CD40, CD80, and CD86, and secreted high levels of IL-12 and IL-18, but not IL-10. [76]

Infants with atopic dermatitis who received hydrolyzed whey formula supplemented with L rhamnosus (GG) showed greater clinical improvement than those who received the hydrolyzed formula alone. They also excreted less TNF-α and α-1-antitrypsin in their stool suggesting that the probiotics decreased gut inflammation. [77] Atopic infants treated with extensively hydrolyzed whey-based formula with L rhamnosus (GG) or B lactis showed greater improvement in severity of skin manifestations than with hydrolysate formula alone. The probiotic-supplemented group also demonstrated a reduction in serum soluble CD4 (a marker of T-cell activation) and an increase in serum TGF-β1 involved in suppressing the inflammatory response via IgE production and oral tolerance induction. [21] These studies suggest that regular probiotic supplementation may stabilize intestinal barrier function and play a role in modulating allergic responses leading to a decreased severity of atopic symptoms, particularly atopic dermatitis associated with cow's milk protein [21,29,78].

A marked anti-inflammatory effect was produced by bifidobacteria with an IL-10 induction by dendritic cells and consequent inhibition of Th1 activation with decreased interferon-γ production [79]. In atopic infants supplemented with B lactis a decrease of Bacteroides and E coli in the stool was shown. Most interestingly, serum IgE correlated with E coli counts, and in highly sensitized infants correlated with Bacteroides counts. Thus, certain probiotics seem to influence the gut's allergen-stimulated inflammatory response and provide a barrier effect against antigens that might otherwise ultimately lead to systemic allergic symptoms such as eczema. [37]

Proliferation and growth of beneficial bacteria in the digestive system is being promoted with the use of prebiotics. Prebiotics are generally considered to be safe and they are naturally present in several kinds of food. A food ingredient must fulfill the following criteria to be considered a prebiotic: it should be hydrolyzed or absorbed in the upper part of the gastrointestinal tract, it has to be a selective substrate for beneficial bacteria in the colon for example bifidobacteria, and it must be able to alter the intestinal microflora towards a healthier composition [80].

In regards to the immunomodulatory effect of prebiotics, the proposed mechanisms of action are the following: They are thought to stimulate the activity of lactic acid bacteria, such as lactobacilli and bifidobacteria, which have immunomodulatory qualities. A second mechanism of action is that fermentation of prebiotics by lactic acid bacteria enhances Short Chain Fatty Acids (SCFA) that they act as energy substrate for colonocytes. It has been shown that SCFA stimulate Interferon-γ and IL-10 production. [81-84]

The immunomodulatory effect of prebiotics on the prevention of atopic dermatitis has been evaluated by several studies. A study by Moro et al showed that a mixture of prebiotic oligosaccharides reduces the incidence of atopic dermatitis during the first six months of age [85]. A study by van der Aa et al determined the effect of Bifidobacterium breve M-16V combined with a prebiotic oligosaccharide mixture (synbiotic) on atopic markers. The synbiotic mixture had no detectable effect on plasma levels of the analysed atopic disease markers in vivo [86]. Another study by de Kivit S, et al investigated the effect of prebiotic galacto- and fructo-oligosaccharides (scGOS/lcFOS) in combination with Bifidobacterium breve M-16V (GF/Bb) on atopy. The study showed that dietary supplementation with GF/Bb enhances serum galectin-9 levels, which associates with the prevention of the allergic symptoms. [87]

In conclusion, although theoretically pro-, pre and synbiotics are promising candidates to prevent or treat AD, results of the clinical trials performed so far are not conclusive. Prevention trials show promising but heterogenic results. Therefore at this moment there is not enough evidence to support the use of pro-, pre-, or synbiotics for prevention of AD in clinical practice. Results of treatment trials are not very convincing, however pro- or synbiotics could possibly play a role in the treatment of IgE-associated AD, which should be elucidated in future prospective trials.

#### **4.5. Necrotizing enterocololitis**

316 Probiotics

RCTs.

**4.4. Allergy**

IL-10. [76]

protein [21,29,78].

and mucosal immunoglobulin A production [75].

and S. thermophilus in sick infants admitted to the hospital, but no such benefit has been identified in healthy children in residential care settings. The already mentioned studies suggest that there is currently not enough evidence to recommend the routine use of probiotics to prevent nosocomial diarrhea. There is a need for large and well-designed

The rationale for using probiotics in prevention and treatment of allergic disorders is based on the concept that appropriate microbial stimuli are required for normal early immunological development. Microbial-gut interactions can improve the integrity of the gut barrier by decreasing intestinal permeability, reducing both adherence of potential antigens and their systemic effect, and by modulating GALT immune response toward antigen tolerance. The intestinal microflora interacts with the mucosal immune system. It has been found that different strains of commercial bacteria vary in the cytokine response they generate. The Th1/Th2 imbalance is crucial to the clinical expression of allergy. Probiotic bacteria can produce significant antiallergenic effects by intricate interactions inducing Th1 cytokines, such as interferon-γ [73] , Τ-regulatory cytokines, such as IL-10 and TGF-β [74] ,

Three species of Lactobacillus were shown to modulate the phenotype and functions of human myeloid dendritic cells (DCs). Lactobacillus-exposed myeloid DCs up-regulated HLA-DR, CD83, CD40, CD80, and CD86, and secreted high levels of IL-12 and IL-18, but not

Infants with atopic dermatitis who received hydrolyzed whey formula supplemented with L rhamnosus (GG) showed greater clinical improvement than those who received the hydrolyzed formula alone. They also excreted less TNF-α and α-1-antitrypsin in their stool suggesting that the probiotics decreased gut inflammation. [77] Atopic infants treated with extensively hydrolyzed whey-based formula with L rhamnosus (GG) or B lactis showed greater improvement in severity of skin manifestations than with hydrolysate formula alone. The probiotic-supplemented group also demonstrated a reduction in serum soluble CD4 (a marker of T-cell activation) and an increase in serum TGF-β1 involved in suppressing the inflammatory response via IgE production and oral tolerance induction. [21] These studies suggest that regular probiotic supplementation may stabilize intestinal barrier function and play a role in modulating allergic responses leading to a decreased severity of atopic symptoms, particularly atopic dermatitis associated with cow's milk

A marked anti-inflammatory effect was produced by bifidobacteria with an IL-10 induction by dendritic cells and consequent inhibition of Th1 activation with decreased interferon-γ production [79]. In atopic infants supplemented with B lactis a decrease of Bacteroides and E coli in the stool was shown. Most interestingly, serum IgE correlated with E coli counts, and in highly sensitized infants correlated with Bacteroides counts. Thus, certain probiotics seem to influence the gut's allergen-stimulated inflammatory response and provide a barrier Microflora establishment and composition in premature infants is a major determinant in the pathophysiology of NEC. The premature infant is exposed to a variety of factors that negatively affect their possibilities of attaining an appropriate colonization. These factors include increasing exposure to potential delayed colonization, colonization with "neonatal intensive care unit environmental microbes", use of antibiotics, lack of exposure to maternal flora and breast milk.

Mechanisms by which probiotics could prevent NEC include increase in favorable type microflora with reduced colonization by pathogens, increased intestinal barrier to translocation of bacteria into the bloodstream, modification of the host response to microbial products by sensitization and immunization, and enhanced tolerance and advancement of enteral nutrition [88-91. ]

Probiotics in Pediatrics – Properties, Mechanisms of Action, and Indications 319

associated with any pathologic or adverse event. Studies so far have documented safety and adequate growth with B. lactis in infants from birth [39] and in vulnerable populations, including preterm infants, [33, 19] malnourished infants, [98] and infants born to mothers

From the safety point of view, according to current available information, Bifidobacteria, particularly B lactis, has a uniquely strong safety profile, making it a good probiotic candidate for newborns and young infants. Lactobacilli, particularly L rhamnosus (GG), also seems generally safe and be appropriate for older infants and children. Until adequate data are available for each specific probiotic bacterium, use of probiotics in general cannot be recommended in immunocompromised populations. However, as safety is better documented for specific bacteria, we may be able to use them in certain populations that

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[2] Maldonado J, Caňabade F,Sempere L,et al. Human milk probiotic Lactobacillus fermentum CECT 5716 reduces the incidence of gastrointestinal and upper respiratory

[3] Corrȇa N, Penna F,Lima F,et al. Treatment of acute diarrhea with Saccharomyces

[4] Food and Agriculture Organization, World Health Organization. The Food and Agriculture Organization of the United Nations and the World Health Organization Joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. FAO/WHO Report No. 10-1-2001. [5] Saavedra JM. Clinical applications of probiotic agents. Am J Clin Nutr. 2001; 73: 1147S-

[7] Harmsen HJ,Wildeboer-Veloo AC,Raangs GC et al. Analysis of intestinal flora development in breast-fed infants and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr. 2000; 30:61-67. [8] Isolauri E,Sutas Y,Kankaanpaa P,Arvilommi H,Salminen S. Probiotics :effects on

[9] Saavedra JM. Use of Probiotics in Pediatrics : Rationale ,Mechanisms of Action ,and

[10] Servin AL. Antagonistic activities of Lactobacilli and Bifidobacteria against microbial

[11] Bakker-Zierikzee AM, Tol EA ,Kroes H ,Alles MS ,Kok FJ ,Bindels JG. Faecal s IgA secretion in infants fed on pre- or probiotic infant formula. Pediatr Allergy Immunol. 2006; 17:134-140.

with HIV disease [99]

**Author details**

Antigoni Mavroudi

**6. References**

1151S.

may benefit the most from probiotic use.

*Aristotle University of Thessaloniki, Greece*

Several RCTs have assessed the efficacy of probiotics in preventing NEC. In a preospective, double-blind study premature infants (n=585) were randomized to receive standard milk formula supplemented with L rhamnosus G, or placebo. The group supplemented with L rhamnosus GG was found to have lower incidence of urinary tract infections and lower, but not statistically significant, incidence of NEC [92]. Two other trials have shown various degrees of reduction in relative risk of NEC with probiotics. The first study compared the incidence of NEC and the mortality of very-low-birth-weight (VLBW) infants fed breast milk with or without added probiotics. Infants (n=187) were randomized to receive breast milk or breast milk with L. acidophilus and B. infantis. In the intervention group the incidence of NEC was significantly decreased compared with the incidence in infants given breast milk alone [93]. The second study compared neonates receiving B infantis, S thermophilus, and B bifidus with neonates receiving no probiotic supplement. The incidence of NEC was 4% in 72 supplemented infants versus 16, 4% in 73 controls. The severity of NEC was less severe in the probiotic group. Three of 15 infants with NEC died, all in the control group [94].

A meta-analysis of RCTs evaluated if probiotic supplementation in preterm (<34 weeks gestation) VLBW(< 1500 gr) neonates could prevent NEC. The risk for NEC and death was significantly lower in the intervention group, while the risk for sepsis was not significantly different between the intervention group and the placebo. No significant adverse effects were reported [95].

In conclusion, specific clinical benefits are increasingly demonstrated for specific bacteria, which determine their probiotic capability. The protective and immune modulatory mechanisms that explain these effects are increasingly being documented.

### **5. Safety concerns of probiotics use**

Newborn infants can develop infection from many species of resident microflora. The mechanisms for these infections and route of contamination are unclear. Many strains of Lactobacilli and Bifidobacteria are generally recognized as safe for use in the food supply. Documented correlations between systemic infections and probiotic consumption are few, and they have all occurred in patients with underlying medical conditions. Sporadic lactobacillemia from environmental, dietary, or fecal lactobacilli has been very rarely reported. Case reports of L rhamnosus (GG) infections possibly associated with probiotic consumption, in immunocompromised patients have been even less common [96, 97].

As opposed to the rarely reported episodes of lactobacillemia (some associated to ingested Lactobacilli), bifidobacteremia has not been sporadically reported, whether associated with consumption of commercial products containing Bifidobacteria or not. Bifidobacteria have also been consumed in infant formulas for more than 15 years worldwide and have not been associated with any pathologic or adverse event. Studies so far have documented safety and adequate growth with B. lactis in infants from birth [39] and in vulnerable populations, including preterm infants, [33, 19] malnourished infants, [98] and infants born to mothers with HIV disease [99]

From the safety point of view, according to current available information, Bifidobacteria, particularly B lactis, has a uniquely strong safety profile, making it a good probiotic candidate for newborns and young infants. Lactobacilli, particularly L rhamnosus (GG), also seems generally safe and be appropriate for older infants and children. Until adequate data are available for each specific probiotic bacterium, use of probiotics in general cannot be recommended in immunocompromised populations. However, as safety is better documented for specific bacteria, we may be able to use them in certain populations that may benefit the most from probiotic use.

## **Author details**

318 Probiotics

enteral nutrition [88-91. ]

were reported [95].

Mechanisms by which probiotics could prevent NEC include increase in favorable type microflora with reduced colonization by pathogens, increased intestinal barrier to translocation of bacteria into the bloodstream, modification of the host response to microbial products by sensitization and immunization, and enhanced tolerance and advancement of

Several RCTs have assessed the efficacy of probiotics in preventing NEC. In a preospective, double-blind study premature infants (n=585) were randomized to receive standard milk formula supplemented with L rhamnosus G, or placebo. The group supplemented with L rhamnosus GG was found to have lower incidence of urinary tract infections and lower, but not statistically significant, incidence of NEC [92]. Two other trials have shown various degrees of reduction in relative risk of NEC with probiotics. The first study compared the incidence of NEC and the mortality of very-low-birth-weight (VLBW) infants fed breast milk with or without added probiotics. Infants (n=187) were randomized to receive breast milk or breast milk with L. acidophilus and B. infantis. In the intervention group the incidence of NEC was significantly decreased compared with the incidence in infants given breast milk alone [93]. The second study compared neonates receiving B infantis, S thermophilus, and B bifidus with neonates receiving no probiotic supplement. The incidence of NEC was 4% in 72 supplemented infants versus 16, 4% in 73 controls. The severity of NEC was less severe in the

probiotic group. Three of 15 infants with NEC died, all in the control group [94].

mechanisms that explain these effects are increasingly being documented.

**5. Safety concerns of probiotics use**

A meta-analysis of RCTs evaluated if probiotic supplementation in preterm (<34 weeks gestation) VLBW(< 1500 gr) neonates could prevent NEC. The risk for NEC and death was significantly lower in the intervention group, while the risk for sepsis was not significantly different between the intervention group and the placebo. No significant adverse effects

In conclusion, specific clinical benefits are increasingly demonstrated for specific bacteria, which determine their probiotic capability. The protective and immune modulatory

Newborn infants can develop infection from many species of resident microflora. The mechanisms for these infections and route of contamination are unclear. Many strains of Lactobacilli and Bifidobacteria are generally recognized as safe for use in the food supply. Documented correlations between systemic infections and probiotic consumption are few, and they have all occurred in patients with underlying medical conditions. Sporadic lactobacillemia from environmental, dietary, or fecal lactobacilli has been very rarely reported. Case reports of L rhamnosus (GG) infections possibly associated with probiotic consumption, in immunocompromised patients have been even less common [96, 97].

As opposed to the rarely reported episodes of lactobacillemia (some associated to ingested Lactobacilli), bifidobacteremia has not been sporadically reported, whether associated with consumption of commercial products containing Bifidobacteria or not. Bifidobacteria have also been consumed in infant formulas for more than 15 years worldwide and have not been Antigoni Mavroudi *Aristotle University of Thessaloniki, Greece*

#### **6. References**

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**Chapter 14**

© 2012 Al-Salami et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Al-Salami et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Probiotics Applications in Autoimmune Diseases**

An autoimmune disorder (AD) is a condition in which the immune system mistakenly attacks its own body cells through the production of antibodies that target certain tissues. Such attack triggers further inflammation that result in more attacks and a significant inflammatory response leading to tissue destruction and cessation of functionality [1]. ADs include diabetes, rheumatoid arthritis, Graves' disease, systemic lupus and inflammatory bowel disease (IBD) [2]. ADs are on the rise worldwide and have major health implications from the diseases themselves as well as complications. Even though the causes of AD have been postulated to be genetic and environmental, the actual triggers remain poorly defined [3]. Genetic predisposition contribute to about 30% of AD while 70% to environmental factors such as infections (e.g., virus, bacteria) and lifestyle-associated factors such as food.