# Evaluation of Cereals and Pseudocereals Suitability for the Development of New Probiotic Foods

Authors: Monika Kocková; Monika Dilongová; Eva Hybenová; L’ubomír Valík
Journal: Journal of Chemistry
 (2013)

---

## Abstract

The aim of the present work was to evaluate suitability of cereals and pseudocereals for the development of new probiotic foods and to evaluate the stability of cereal and pseudocereal porridges fermented by probiotic strainLactobacillus rhamnosus GG. Ten samples of cereals and pseudocereals obtained from Slovak mill house and markets were used in this work. A mixture of each cereal and pseudocereal samples with water (10% w/v) was inoculated after sterilization with coequal number of Lactobacillus rhamnosus GG, to obtain approximately 5-6 log colony form units per gram of suspensions. Fermentation was led at 37°C during 10 hours. Fermented suspensions were stored for 21 days at 5°C. Monitoring of cell counts, pH value, and concentration of organic acids during fermentation and storage was done.


---

## Body

## 1. Introduction

Cereals and pseudocereals belong to the most important food for the majority of mankind. They are a good source of saccharides (especially starch and fibres), proteins (with good quality of amino acids, except lysine in case of cereal grains), lipids (essential fatty acids, almost no presence of saturated fatty acids), vitamins (B group), and minerals (calcium, potassium, magnesium, ferrum, zinc, cuprum, and phosphorus) [1, 2]. On the other hand, antinutritive factors, such as phytic acid, tannins, polyphenols, and enzyme inhibitors, that decrease nutritional quality of grains by binding proteins, saccharides and mineral to complexes are naturally present in cereal and pseudocereal grains [3].Cereals and pseudocereals are usually consumed in the form of bread, breakfast cereals, or cereal bars in developed countries. On the other hand, in developing countries the consumption of fermented cereals in the form of beverages, cakes, or porridges is common. These kinds of food would increase consumption of cereals and mainly pseudocereals in developed countries and moreover contribute to increase probiotic intake, in case of using probiotic strain for fermentation process [4].Fermentation conduces to the enhancement of nutritional quality of end products via production of nutritive factors (e.g., biogenic amines andγ-aminobutyric acid) and reduction of antinutritive factors [3, 5]. Fermentation also makes for improvement of sensory quality of end products in comparison to raw materials by producing aroma compounds by lactic acid bacteria [3, 6] and to the extension of shelf life, through the production of antimicrobial compounds [7, 8].Probiotics are live microorganisms which have a positive effect on consumer [9]. Lactobacillus rhamnosus GG is one of the most monitored probiotic strains. It belongs to Gram-positive, non-spore-forming, nonmotile, catalase-negative, facultatively anaerobic or microaerophilic, and mesophilic bacteria [10]. Metabolism of Lb. rhamnosus GG is facultatively heterofermentative; it can produce acetic acid, formic acid and ethanol, in addition to lactic acid, in lack of glucose in fermentation environment [11]. Lb. rhamnosus GG is highly tolerant to acidic environment of the stomach; it is able to survive in intestinal passage, adhere to intestinal mucosa, and colonize gastrointestinal tract after three days of consumption [12, 13]. It enhances human natural resistance and healthy digestive system and inhibits adhesion of some pathogenic bacteria. It relieves syndromes of irritation of GI tract, atopic dermatitis, and cow milk allergy [14, 15].In Slovak food market, probiotic foods are mainly milk based, and therefore probiotics are not available for all consumers, especially for those who suffer from milk allergies and intolerances. This was the reason why we focused our research on the selection of plant materials (cereals, pseudocereals, and legumes) on the bases of growth and metabolic characteristics of selected probiotic strain,Lactobacillus rhamnosus GG, which will lead to the development of new probiotic foods.


## 2. Materials and Methods

### 2.1. Starter Culture

The probiotic strain ofLb. rhamnosus GG was used in this work. It was provided by Dr. Salminen (University of Turku, Turku, Finland) through mediation of Dr. Lauková (State Veterinary and Food Institute, Košice, Slovakia).


### 2.2. Cereal and Pseudocereal Substrates

Ten samples of cereals and pseudocereals were used in this work. Rye flour (RF), rye grain (RG), barley flour (BF), and whole barley flour (WBF), amaranth flour (AF), buckwheat flour (BWF), whole buckwheat flour (WBWF), whole oat flour (WOF) were obtained from mill house (Mlyn Zrno, Šíšov, Slovak Republic); amaranth grain (AG, Primeal, Peaugres, France) and millet grain (MG, Marianna wholesale, Ivánka pri Dunaji, Slovak Republic) were obtained from market. Sixty grams of flours or milled and sieved grains were mixed with 540 mL of deionised water for preparation of fermentation substrates.


### 2.3. Fermentation and Storage

Substrates were inoculated after autoclaving for 15 min at 121°C and cooling down with overnight culture (MRS broth, 18 hours, 37°C) ofLb. rhamnosus GG to give approximately 5 to 6 log colony form units per gram of suspensions. Static fermentation was performed for 10 hours (according to the previous results) at 37°C. Samples for analyses were taken every 2 hours. Storage observations were carried out at 5°C for 21 days. Samples for analyses were taken every 2-3 days.


### 2.4. Viable Cell Enumeration

Enumeration of viable cells ofLb. rhamnosus GG was performed by estimation of colony forming unit number on MRS-agar plates according to the STN ISO 15214.


### 2.5.  Determination of pH and Organic Acids

The pH of samples was measured by pH-meter CG 843 (Schott, Mainz, Germany). The quality and quantity of the produced organic acids were measured by isotachophoretic analysis by using the Isotachophoretic Analyser ZKI 01 (Villa Labeco, Spišská Nová Ves, Slovak Republic). Electrolytic system according to Kocková et al. [16] was used. Quantitative analysis was performed by calibration of standard solution of lactic, acetic, citric, formic, and succinic acids (Lachema, Brno, Czech Republic).


### 2.6. Estimation of Growth and Metabolic Parameters ofLb. rhamnosus GG

Growth curves ofLb. rhamnosusGG in each substrate were modelled with a mechanistic model of Baranyi and Roberts [17]. Growth and metabolic parameters were calculated from each curve.


### 2.7. Statistical Analyses

Each experiment was performed in three separate trials. Results represented means with standard deviations. Statistical analyses were carried out using Microsoft Excel 2007 (Microsoft, Redmond, WA, USA). Data were treated by ANOVA test with a least significant difference of 95%.


## 2.1. Starter Culture

The probiotic strain ofLb. rhamnosus GG was used in this work. It was provided by Dr. Salminen (University of Turku, Turku, Finland) through mediation of Dr. Lauková (State Veterinary and Food Institute, Košice, Slovakia).


## 2.2. Cereal and Pseudocereal Substrates

Ten samples of cereals and pseudocereals were used in this work. Rye flour (RF), rye grain (RG), barley flour (BF), and whole barley flour (WBF), amaranth flour (AF), buckwheat flour (BWF), whole buckwheat flour (WBWF), whole oat flour (WOF) were obtained from mill house (Mlyn Zrno, Šíšov, Slovak Republic); amaranth grain (AG, Primeal, Peaugres, France) and millet grain (MG, Marianna wholesale, Ivánka pri Dunaji, Slovak Republic) were obtained from market. Sixty grams of flours or milled and sieved grains were mixed with 540 mL of deionised water for preparation of fermentation substrates.


## 2.3. Fermentation and Storage

Substrates were inoculated after autoclaving for 15 min at 121°C and cooling down with overnight culture (MRS broth, 18 hours, 37°C) ofLb. rhamnosus GG to give approximately 5 to 6 log colony form units per gram of suspensions. Static fermentation was performed for 10 hours (according to the previous results) at 37°C. Samples for analyses were taken every 2 hours. Storage observations were carried out at 5°C for 21 days. Samples for analyses were taken every 2-3 days.


## 2.4. Viable Cell Enumeration

Enumeration of viable cells ofLb. rhamnosus GG was performed by estimation of colony forming unit number on MRS-agar plates according to the STN ISO 15214.


## 2.5.  Determination of pH and Organic Acids

The pH of samples was measured by pH-meter CG 843 (Schott, Mainz, Germany). The quality and quantity of the produced organic acids were measured by isotachophoretic analysis by using the Isotachophoretic Analyser ZKI 01 (Villa Labeco, Spišská Nová Ves, Slovak Republic). Electrolytic system according to Kocková et al. [16] was used. Quantitative analysis was performed by calibration of standard solution of lactic, acetic, citric, formic, and succinic acids (Lachema, Brno, Czech Republic).


## 2.6. Estimation of Growth and Metabolic Parameters ofLb. rhamnosus GG

Growth curves ofLb. rhamnosusGG in each substrate were modelled with a mechanistic model of Baranyi and Roberts [17]. Growth and metabolic parameters were calculated from each curve.


## 2.7. Statistical Analyses

Each experiment was performed in three separate trials. Results represented means with standard deviations. Statistical analyses were carried out using Microsoft Excel 2007 (Microsoft, Redmond, WA, USA). Data were treated by ANOVA test with a least significant difference of 95%.


## 3. Results and Discussion

### 3.1. Fermentation

Fermentation of cereal and pseudocereal substrates for evaluation of their suitability for lactic acid fermentation was carried out. Cell counts, pH value, and organic acid concentrations were monitored in fixed period. Growth and metabolic curves are shown in Figure1 and growth and metabolic parameters in Tables 1–5.Table 1
Growth parameters ofLb. rhamnosus GG during fermentation of cereal and pseudocereal substrates.
Substrate
Gr [log cfu g−1 h−1]
t
d [h]
λ [h]
N
0 [log cfu g−1]
N
max [log cfu g−1]
RF
0.297b
1.01d
1.64c
6.20f
8.57f
RG
0.372c
0.81c
0.73a
5.05a
7.78c
BF
0.287b
1.05d
2.44d
6.42g
8.41e
WBF
0.430d
0.70b
1.42b
5.59e
7.65b
AF
0.589e
0.51a
3.88e
5.23b
8.80g
AG
0.319b
0.94c
1.79c
5.36c
7.96d
BWF
0.324b
0.93c
2.53d
5.36c
7.67b
WBWF
0.328b
0.92c
2.68d
5.48d
7.81c
WOF
0.248a
1.21d
1.42b
5.45d
7.40a
MG
0.263a
1.15d
—
5.06a
7.67b
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
Gr: growth rate,td: time to double, λ: lag phase, N0: initial density of Lb. rhamnosus GG, Nmax: final density of Lb. rhamnosus GG.
a–gMeans within a column with different superscript letters are significantly different (P< 0.05).Table 2
Parameters of pH value changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
pH [h−1]
λ
pH [h]
pH0
pHend
RF
−0.083f
—
5.35b
4.62b
RG
−0.029d
—
5.87d
5.60e
BF
−0.047e
—
5.00a
4.78b
WBF
−0.026d
—
5.84d
5.59e
AF
−0.008b
—
6.10e
5.99f
AG
−0.355g
7.90b
6.00e
5.24d
BWF
−1.063h
8.70c
5.88d
4.31a
WBWF
−0.003a
—
5.63c
5.59e
WOF
−0.013c
—
5.10a
4.91c
MG
−0.244g
6.50a
5.91d
5.06c
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
pH: rate of changes in pH values, λpH: lag phase of pH changes, pH0: initial pH, pHend: final pH.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).Table 3
Parameters of lactic acid concentration changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
acid [mg kg−1 h−1]
λ
acid [h]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
53.07d
1.58a
11.27b
349.98d
RG
24.31a
2.50b
0.00a
221.16b
BF
78.81e
3.12c
52.51c
529.31f
WBF
192.85g
7.43e
64.14d
559.60g
AF
40.42c
8.30g
136.40g
205.15b
AG
195.91g
7.35e
124.09f
644.21h
BWF
152.95f
7.61f
0.00a
391.14e
WBWF
53.22d
7.38e
81.05e
220.40b
WOF
32.02b
5.00d
0.00a
174.66a
MG
51.83d
4.80d
0.00a
271.47c
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of lactic acid concentration changes, λacid: lag phase of lactic acid concentration changes, c0: initial concentration of lactic acid, cend: final concentration of lactic acid.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).Table 4
Parameters of acetic acid concentration changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
acid [mg kg−1 h−1]
λ
acid [h]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
4.34f
—
35.44b
76.98c
RG
30.59h
—
0.50a
79.01c
BF
18.64g
6.28b
56.93d
126.06e
WBF
0.29c
—
65.29e
72.14c
AF
16.92g
3.95a
112.77g
221.21f
AG
−3.99a
—
147.53h
94.87d
BWF
—
—
—
—
WBWF
−1.99b
—
69.12e
34.61a
WOF
0.97d
—
77.35f
90.64d
MG
1.56e
—
44.68c
57.87b
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of acetic acid concentration changes, λacid: lag phase of acetic acid concentration changes, c0: initial concentration of acetic acid, cend: final concentration of acetic acid.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).Table 5
Parameters of citric acid concentration changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
acid [mg kg−1 h−1]
λ
acid [h]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
−24.36g
3.90a
295.49d
153.70c
RG
−3.06b
—
223.86c
161.56c
BF
−47.79i
3.88a
525.03e
278.35e
WBF
−6.80e
—
543.68f
484.96f
AF
−8.02e
—
1140.13h
1028.97h
AG
−15.40f
—
696.34g
496.19f
BWF
7.43a
—
76.71a
140.23b
WBWF
−5.97d
—
657.78g
559.08g
WOF
−4.49c
—
167.64b
68.36a
MG
−37.40h
7.91b
291.26d
213.22d
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of citric acid concentration changes, λacid: lag phase of citric acid concentration changes, c0: initial concentration of citric acid, cend: final concentration of citric acid.
a–iMeans within a column with different superscript letters are significantly different (P< 0.05).Evaluation of cell counts ofLb. rhamnosus GG (a) and changes in pH values (b) and concentration of lactic acid (c), acetic acid (d), and citric acid (e) during fermentation.
(a)
(b)
(c)
(d)
(e)As it is shown in Figure1, probiotic strain Lb. rhamnosus GG was able to grow and metabolize in each cereal and pseudocereal substrate during a ten-hour fermentation process. Lb. rhamnosus GG was able to grow from initial cell counts 5.04–6.46 log cfu g−1 to final 7.40–8.80 log cfu g−1 (Table 1), which is similar to density of Lb. rhamnosus GG reached in milk [10], in cereal water-based puddings [18], and maize porridges with barley addition [19]. The highest growth rate was calculated in case of amaranth flour (in which the longest lag phase was observed), 0.589 log cfu g−1 h−1. The lowest growth rate was calculated for whole oat flour, 0.248 log cfu g−1 h−1 (Table 1). Growth rate of Lb. rhamnosus GG during fermentation of milk at 35°C was 0.653 log cfu mL−1 h−1 [10]. Higher growth rate was found during fermentation of amaranth milk- and water-based puddings and buckwheat milk-based pudding by Lb. rhamnosus GG [20]. Time to double ranged from 0.51 h (amaranth flour) to 1.21 h (whole oat flour). Lag phase, time for adapting microorganisms to new environment, ranged from 0.73 h (rye grain) to 3.88 h (amaranth flour). In substrate from millet grain, no lag phase was observed (Table 1).During fermentation, organic acids production, as a result of metabolic activity ofLb. rhamnosus GG, was observed. This caused decreasing of pH values in each substrate. pH values dropped from initial 4.93–6.08 to final 4.31–5.99 (Figure 1, Table 2). Final pH value caused by metabolic activity of Lb. rhamnosus GG in MRS broth was 4.00 [21], in milk 6.50 [10] and in milk-based cereal puddings and maize porridges with barley under 4.00 [18, 19]. In case of substrates from amaranth grain, buckwheat flour, and millet grain, a long lag phase of reducing pH values (7.90, 8.70, and 6.50 h, resp.) was observed. In these substrates, higher rate of reducing pH (−0.355, −1.063, and −0.244 h−1, resp.) was calculated. Rates of decreasing pH values in other substrates ranged from −0.003 h−1 (whole buckwheat flour) to −0.047 h−1 (barley flour) (Table 2). In amaranth and buckwheat water- and milk-based pudding rates of pH value decreasing ranged from 0.144 to 0.317 h−1 [20].The concentration of lactic acid at the beginning of fermentation process was established from 11.27 mg kg−1 in case of rye flour to 136.40 mg kg−1 in case of amaranth flour. In substrates from rye grain, buckwheat flour, whole oat flour, and millet grain concentration of lactic acid was under detection limit. The increase in lactic acid concentration was observed in all substrates (Figure 1, Table 3). The highest rate of production of lactic acid was calculated for whole barley flour and amaranth grain, 192.85 and 195.91 mg kg−1 h−1, respectively. In these substrates, long lag phase of lactic acid production was observed, 7.43 and 7.35 h, respectively. Long lag phase was observed also in case of amaranth flour, buckwheat flour and whole buckwheat flour, ranged from 7.38 to 8.30 h. The slowest lactic acid production was calculated in substrate from rye grain, 24.31 mg kg−1 h−1. Lactic acid level at the end of fermentation process ranged from 174.66 mg kg−1 (whole oat flour) to 644.21 mg kg−1 (amaranth grain) (Table 3). Lactic acid concentration after 18 hours fermentation by LAB in MRS broth was 36 g L−1 and in milk lower than 900 mg L−1 [21]. In maize porridges fermented by Lb. rhamnosus GG, lactic acid concentrations were 3500 mg kg−1 and 4000 mg kg−1, respectively [19]. Lactic acid level in cereal water-based pudding fermented by Lb. rhamnosus GG was 2600 mg kg−1 and in milk-based 9800 mg kg−1 [18]. Rates of production of lactic acid in buckwheat and amaranth water-based puddings during 8 hours fermentation were 162 mg L−1 h−1 and 280 mg L−1 h−1, respectively [20].The rate of acetic acid production was slower compared to rate of lactic acid production, ranged from 0.29 mg kg−1 h−1 (whole barley flour) to 30.59 mg kg−1 h−1 (rye grain), which was good for final sensory quality of substrates. In case of amaranth grain and whole buckwheat flour, reduction in acetic acid level was observed and in substrate from buckwheat flour concentration of acetic acid was under detection limit during process (Figure 1, Table 4). Generally, production of acetic acid in our substrates was lower in compare to production of acetic acid in MRS broth or milk by the same probiotic strain [21].Reducing in citric acid level was observed in all substrates, except those prepared from buckwheat flour. In case of rye flour, barley flour and millet grain, lag phase of reducing citric acid was observed and calculated, what caused higher rate of reducing citric acid, ranged from −24.36 to −47.79 mg kg−1 h−1. In other substrates, rate of reducing citric acid ranged from −3.06 (rye grain) to −15.40 mg kg−1 h−1 (amaranth grain) (Figure 1, Table 5). Partial utilization of citric acid by Lb. rhamnosus GG was observed in maize porridges [19] and also in cereal water- and milk-based puddings [18].Only small insignificant (P>0.05) changes in formic and succinic acids content during fermentation were observed in all substrates (results are no shown), which concurs with the results of Helland et al. [19].


### 3.2. Storage

During storage at refrigerated temperature, changes in cell counts and metabolic activity were evaluated. For probiotic food, it is necessary to contain probiotic strain at certain level (6 log cfu per gram or millilitre of products, at least), and any metabolic activity of present microorganism is important.As shown in Figure2, density of Lb. rhamnosus GG after 21 days of storage was over the limit required for probiotic food, except substrate prepared from whole buckwheat flour, in which cell counts of Lb. rhamnosus GG decreased with growth rate −0.00517 log cfu g−1 h−1. Visible reduction in density of Lb. rhamnosus GG was observed also in case of rye, barley, and amaranth flours (Figure 2, Table 6).Table 6
Growth parameters ofLb. rhamnosus GG during storage of fermented cereal and pseudocereal substrates.
Substrate
Gr [log cfu g−1 h−1]
N
0 [log cfu g−1]
N
max [log cfu g−1]
RF
−0,00154d
8,29e
7,49d
RG
0,00019i
7,66b
7,93g
BF
−0,00300b
8,59f
7,15c
WBF
0,00024j
8,06d
7,69e
AF
−0,00188c
7,96d
6,70b
AG
−0,00031h
8,02d
7,79f
BWF
−0,00076e
7,91d
7,46d
WBWF
−0,00517a
7,80c
5,85a
WOF
−0,00037g
7,35a
7,41d
MG
−0,00048f
7,83c
7,60e
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
Gr: growth rate,N0: initial density of Lb. rhamnosus GG, Nmax: final density of Lb. rhamnosus GG.
a–jMeans within a column with different superscript letters are significantly different (P< 0.05).Evaluation of cell counts ofLb. rhamnosus GG (a) and changes in pH values (b), and concentration of lactic acid (c), acetic acid (d) and citric acid (e) during storage of fermented cereal and pseudocereal substrates.
(a)
(b)
(c)
(d)
(e)All fermented porridges were metabolically stabile except those prepared from amaranth flour, in which production of lactic acid (3.752 mg kg−1 h−1) and reduction of citric acid (−0.732 mg kg−1 h−1) and pH values (−0.00435 h−1) continued, and from whole buckwheat flour, in which metabolic activity continued during first five days of the storage period (Figure 2, Tables 7, 8, 9, and 10). According to these results, we evaluated used substrates as metabolically stabile, except substrate prepared from amaranth flour, in which higher content of saccharides, proteins, and lipids were found [16]. Evaluation of stability and definition of shelf-life of fermented cereal products was done only in scarce research. Angelov et al. [22] estimated shelf-life of probiotic oat drink to 21 days as a result of 24-day observation during storage period.Table 7
Parameters of pH changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
pH [h−1]
pH0
pHend
RF
0,00033g
4,89b
5,22c
RG
0,00018f
5,34d
5,47d
BF
0,00005e
4,78b
4,82b
WBF
−0,00061b
5,38d
5,13c
AF
−0,00435a
6,17e
4,52a
AG
−0,00077b
5,48d
5,39c
BWF
−0,00016c
4,58a
4,49a
WBWF
−0,00677a
5,50d
4,87b
WOF
−0,00004d
5,12c
5,15c
MG
0,00022f
5,23c
5,30c
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
pH: rate of changes in pH values, pH0: initial pH, pHend: final pH.
a–gMeans within a column with different superscript letters are significantly different (P< 0.05).Table 8
Parameters of lactic acid concentration changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
acid [mg kg−1 h−1]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
−0,041c
276,52a
236,41a
RG
0,126g
404,90c
401,05d
BF
−0,122a
469,20e
361,41c
WBF
−0,060b
617,95f
603,81f
AF
3,752i
402,90c
2217,50i
AG
0,092f
883,81g
925,48g
BWF
−0,011d
395,75c
415,53d
WBWF
4,967j
416,12d
1122,91h
WOF
0,079e
255,74a
279,09b
MG
2,455h
359,89b
483,38e
RF: rye flour, RG: rye grain, BF:  barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of lactic acid concentration changes, c0: initial concentration of lactic acid, cend: final concentration of lactic acid.
a–iMeans within a column with different superscript letters are significantly different (P< 0.05).Table 9
Parameters of acetic acid concentration changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
acid [mg kg−1 h−1]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
0,016d
60,60b
63,15b
RG
0,069f
94,91d
117,07e
BF
0,007c
88,67d
78,48c
WBF
0,058e
94,44d
120,77e
AF
−0,091b
258,30f
227,02f
AG
0,098g
162,46e
225,97f
BWF
6,793h
0,05a
40,87a
WBWF
−0,236a
60,94b
37,78a
WOF
0,052e
92,55d
117,28e
MG
0,015d
82,33c
90,11d
RF: rye flour, RG: rye grain, BF:  barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of acetic acid concentration changes, c0: initial concentration of acetic acid, cend: final concentration of acetic acid.
a–hMeans within a column with different superscript letters are significantly different (P < 0.05).Table 10
Parameters of citric acid concentration changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
acid [mg kg−1 h−1]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
−0,014d
170,02b
155,95a
RG
—
—
—
BF
−0,066f
216,76c
165,06a
WBF
−0,028e
478,37d
470,37d
AF
−0,732g
1121,94f
822,96f
AG
0,120b
515,66d
581,54e
BWF
0,003a
130,93a
136,86a
WBWF
−3,161h
557,95e
429,94c
WOF
—
—
—
MG
0,037c
228,06c
249,15b
RF: rye flour, RG: rye grain, BF:  barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of citric acid concentration changes, c0: initial concentration of citric acid, cend: final concentration of citric acid.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).


## 3.1. Fermentation

Fermentation of cereal and pseudocereal substrates for evaluation of their suitability for lactic acid fermentation was carried out. Cell counts, pH value, and organic acid concentrations were monitored in fixed period. Growth and metabolic curves are shown in Figure1 and growth and metabolic parameters in Tables 1–5.Table 1
Growth parameters ofLb. rhamnosus GG during fermentation of cereal and pseudocereal substrates.
Substrate
Gr [log cfu g−1 h−1]
t
d [h]
λ [h]
N
0 [log cfu g−1]
N
max [log cfu g−1]
RF
0.297b
1.01d
1.64c
6.20f
8.57f
RG
0.372c
0.81c
0.73a
5.05a
7.78c
BF
0.287b
1.05d
2.44d
6.42g
8.41e
WBF
0.430d
0.70b
1.42b
5.59e
7.65b
AF
0.589e
0.51a
3.88e
5.23b
8.80g
AG
0.319b
0.94c
1.79c
5.36c
7.96d
BWF
0.324b
0.93c
2.53d
5.36c
7.67b
WBWF
0.328b
0.92c
2.68d
5.48d
7.81c
WOF
0.248a
1.21d
1.42b
5.45d
7.40a
MG
0.263a
1.15d
—
5.06a
7.67b
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
Gr: growth rate,td: time to double, λ: lag phase, N0: initial density of Lb. rhamnosus GG, Nmax: final density of Lb. rhamnosus GG.
a–gMeans within a column with different superscript letters are significantly different (P< 0.05).Table 2
Parameters of pH value changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
pH [h−1]
λ
pH [h]
pH0
pHend
RF
−0.083f
—
5.35b
4.62b
RG
−0.029d
—
5.87d
5.60e
BF
−0.047e
—
5.00a
4.78b
WBF
−0.026d
—
5.84d
5.59e
AF
−0.008b
—
6.10e
5.99f
AG
−0.355g
7.90b
6.00e
5.24d
BWF
−1.063h
8.70c
5.88d
4.31a
WBWF
−0.003a
—
5.63c
5.59e
WOF
−0.013c
—
5.10a
4.91c
MG
−0.244g
6.50a
5.91d
5.06c
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
pH: rate of changes in pH values, λpH: lag phase of pH changes, pH0: initial pH, pHend: final pH.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).Table 3
Parameters of lactic acid concentration changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
acid [mg kg−1 h−1]
λ
acid [h]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
53.07d
1.58a
11.27b
349.98d
RG
24.31a
2.50b
0.00a
221.16b
BF
78.81e
3.12c
52.51c
529.31f
WBF
192.85g
7.43e
64.14d
559.60g
AF
40.42c
8.30g
136.40g
205.15b
AG
195.91g
7.35e
124.09f
644.21h
BWF
152.95f
7.61f
0.00a
391.14e
WBWF
53.22d
7.38e
81.05e
220.40b
WOF
32.02b
5.00d
0.00a
174.66a
MG
51.83d
4.80d
0.00a
271.47c
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of lactic acid concentration changes, λacid: lag phase of lactic acid concentration changes, c0: initial concentration of lactic acid, cend: final concentration of lactic acid.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).Table 4
Parameters of acetic acid concentration changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
acid [mg kg−1 h−1]
λ
acid [h]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
4.34f
—
35.44b
76.98c
RG
30.59h
—
0.50a
79.01c
BF
18.64g
6.28b
56.93d
126.06e
WBF
0.29c
—
65.29e
72.14c
AF
16.92g
3.95a
112.77g
221.21f
AG
−3.99a
—
147.53h
94.87d
BWF
—
—
—
—
WBWF
−1.99b
—
69.12e
34.61a
WOF
0.97d
—
77.35f
90.64d
MG
1.56e
—
44.68c
57.87b
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of acetic acid concentration changes, λacid: lag phase of acetic acid concentration changes, c0: initial concentration of acetic acid, cend: final concentration of acetic acid.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).Table 5
Parameters of citric acid concentration changes during fermentation of cereal and pseudocereal substrates withLb. rhamnosus GG.
Substrate
k
acid [mg kg−1 h−1]
λ
acid [h]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
−24.36g
3.90a
295.49d
153.70c
RG
−3.06b
—
223.86c
161.56c
BF
−47.79i
3.88a
525.03e
278.35e
WBF
−6.80e
—
543.68f
484.96f
AF
−8.02e
—
1140.13h
1028.97h
AG
−15.40f
—
696.34g
496.19f
BWF
7.43a
—
76.71a
140.23b
WBWF
−5.97d
—
657.78g
559.08g
WOF
−4.49c
—
167.64b
68.36a
MG
−37.40h
7.91b
291.26d
213.22d
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of citric acid concentration changes, λacid: lag phase of citric acid concentration changes, c0: initial concentration of citric acid, cend: final concentration of citric acid.
a–iMeans within a column with different superscript letters are significantly different (P< 0.05).Evaluation of cell counts ofLb. rhamnosus GG (a) and changes in pH values (b) and concentration of lactic acid (c), acetic acid (d), and citric acid (e) during fermentation.
(a)
(b)
(c)
(d)
(e)As it is shown in Figure1, probiotic strain Lb. rhamnosus GG was able to grow and metabolize in each cereal and pseudocereal substrate during a ten-hour fermentation process. Lb. rhamnosus GG was able to grow from initial cell counts 5.04–6.46 log cfu g−1 to final 7.40–8.80 log cfu g−1 (Table 1), which is similar to density of Lb. rhamnosus GG reached in milk [10], in cereal water-based puddings [18], and maize porridges with barley addition [19]. The highest growth rate was calculated in case of amaranth flour (in which the longest lag phase was observed), 0.589 log cfu g−1 h−1. The lowest growth rate was calculated for whole oat flour, 0.248 log cfu g−1 h−1 (Table 1). Growth rate of Lb. rhamnosus GG during fermentation of milk at 35°C was 0.653 log cfu mL−1 h−1 [10]. Higher growth rate was found during fermentation of amaranth milk- and water-based puddings and buckwheat milk-based pudding by Lb. rhamnosus GG [20]. Time to double ranged from 0.51 h (amaranth flour) to 1.21 h (whole oat flour). Lag phase, time for adapting microorganisms to new environment, ranged from 0.73 h (rye grain) to 3.88 h (amaranth flour). In substrate from millet grain, no lag phase was observed (Table 1).During fermentation, organic acids production, as a result of metabolic activity ofLb. rhamnosus GG, was observed. This caused decreasing of pH values in each substrate. pH values dropped from initial 4.93–6.08 to final 4.31–5.99 (Figure 1, Table 2). Final pH value caused by metabolic activity of Lb. rhamnosus GG in MRS broth was 4.00 [21], in milk 6.50 [10] and in milk-based cereal puddings and maize porridges with barley under 4.00 [18, 19]. In case of substrates from amaranth grain, buckwheat flour, and millet grain, a long lag phase of reducing pH values (7.90, 8.70, and 6.50 h, resp.) was observed. In these substrates, higher rate of reducing pH (−0.355, −1.063, and −0.244 h−1, resp.) was calculated. Rates of decreasing pH values in other substrates ranged from −0.003 h−1 (whole buckwheat flour) to −0.047 h−1 (barley flour) (Table 2). In amaranth and buckwheat water- and milk-based pudding rates of pH value decreasing ranged from 0.144 to 0.317 h−1 [20].The concentration of lactic acid at the beginning of fermentation process was established from 11.27 mg kg−1 in case of rye flour to 136.40 mg kg−1 in case of amaranth flour. In substrates from rye grain, buckwheat flour, whole oat flour, and millet grain concentration of lactic acid was under detection limit. The increase in lactic acid concentration was observed in all substrates (Figure 1, Table 3). The highest rate of production of lactic acid was calculated for whole barley flour and amaranth grain, 192.85 and 195.91 mg kg−1 h−1, respectively. In these substrates, long lag phase of lactic acid production was observed, 7.43 and 7.35 h, respectively. Long lag phase was observed also in case of amaranth flour, buckwheat flour and whole buckwheat flour, ranged from 7.38 to 8.30 h. The slowest lactic acid production was calculated in substrate from rye grain, 24.31 mg kg−1 h−1. Lactic acid level at the end of fermentation process ranged from 174.66 mg kg−1 (whole oat flour) to 644.21 mg kg−1 (amaranth grain) (Table 3). Lactic acid concentration after 18 hours fermentation by LAB in MRS broth was 36 g L−1 and in milk lower than 900 mg L−1 [21]. In maize porridges fermented by Lb. rhamnosus GG, lactic acid concentrations were 3500 mg kg−1 and 4000 mg kg−1, respectively [19]. Lactic acid level in cereal water-based pudding fermented by Lb. rhamnosus GG was 2600 mg kg−1 and in milk-based 9800 mg kg−1 [18]. Rates of production of lactic acid in buckwheat and amaranth water-based puddings during 8 hours fermentation were 162 mg L−1 h−1 and 280 mg L−1 h−1, respectively [20].The rate of acetic acid production was slower compared to rate of lactic acid production, ranged from 0.29 mg kg−1 h−1 (whole barley flour) to 30.59 mg kg−1 h−1 (rye grain), which was good for final sensory quality of substrates. In case of amaranth grain and whole buckwheat flour, reduction in acetic acid level was observed and in substrate from buckwheat flour concentration of acetic acid was under detection limit during process (Figure 1, Table 4). Generally, production of acetic acid in our substrates was lower in compare to production of acetic acid in MRS broth or milk by the same probiotic strain [21].Reducing in citric acid level was observed in all substrates, except those prepared from buckwheat flour. In case of rye flour, barley flour and millet grain, lag phase of reducing citric acid was observed and calculated, what caused higher rate of reducing citric acid, ranged from −24.36 to −47.79 mg kg−1 h−1. In other substrates, rate of reducing citric acid ranged from −3.06 (rye grain) to −15.40 mg kg−1 h−1 (amaranth grain) (Figure 1, Table 5). Partial utilization of citric acid by Lb. rhamnosus GG was observed in maize porridges [19] and also in cereal water- and milk-based puddings [18].Only small insignificant (P>0.05) changes in formic and succinic acids content during fermentation were observed in all substrates (results are no shown), which concurs with the results of Helland et al. [19].


## 3.2. Storage

During storage at refrigerated temperature, changes in cell counts and metabolic activity were evaluated. For probiotic food, it is necessary to contain probiotic strain at certain level (6 log cfu per gram or millilitre of products, at least), and any metabolic activity of present microorganism is important.As shown in Figure2, density of Lb. rhamnosus GG after 21 days of storage was over the limit required for probiotic food, except substrate prepared from whole buckwheat flour, in which cell counts of Lb. rhamnosus GG decreased with growth rate −0.00517 log cfu g−1 h−1. Visible reduction in density of Lb. rhamnosus GG was observed also in case of rye, barley, and amaranth flours (Figure 2, Table 6).Table 6
Growth parameters ofLb. rhamnosus GG during storage of fermented cereal and pseudocereal substrates.
Substrate
Gr [log cfu g−1 h−1]
N
0 [log cfu g−1]
N
max [log cfu g−1]
RF
−0,00154d
8,29e
7,49d
RG
0,00019i
7,66b
7,93g
BF
−0,00300b
8,59f
7,15c
WBF
0,00024j
8,06d
7,69e
AF
−0,00188c
7,96d
6,70b
AG
−0,00031h
8,02d
7,79f
BWF
−0,00076e
7,91d
7,46d
WBWF
−0,00517a
7,80c
5,85a
WOF
−0,00037g
7,35a
7,41d
MG
−0,00048f
7,83c
7,60e
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
Gr: growth rate,N0: initial density of Lb. rhamnosus GG, Nmax: final density of Lb. rhamnosus GG.
a–jMeans within a column with different superscript letters are significantly different (P< 0.05).Evaluation of cell counts ofLb. rhamnosus GG (a) and changes in pH values (b), and concentration of lactic acid (c), acetic acid (d) and citric acid (e) during storage of fermented cereal and pseudocereal substrates.
(a)
(b)
(c)
(d)
(e)All fermented porridges were metabolically stabile except those prepared from amaranth flour, in which production of lactic acid (3.752 mg kg−1 h−1) and reduction of citric acid (−0.732 mg kg−1 h−1) and pH values (−0.00435 h−1) continued, and from whole buckwheat flour, in which metabolic activity continued during first five days of the storage period (Figure 2, Tables 7, 8, 9, and 10). According to these results, we evaluated used substrates as metabolically stabile, except substrate prepared from amaranth flour, in which higher content of saccharides, proteins, and lipids were found [16]. Evaluation of stability and definition of shelf-life of fermented cereal products was done only in scarce research. Angelov et al. [22] estimated shelf-life of probiotic oat drink to 21 days as a result of 24-day observation during storage period.Table 7
Parameters of pH changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
pH [h−1]
pH0
pHend
RF
0,00033g
4,89b
5,22c
RG
0,00018f
5,34d
5,47d
BF
0,00005e
4,78b
4,82b
WBF
−0,00061b
5,38d
5,13c
AF
−0,00435a
6,17e
4,52a
AG
−0,00077b
5,48d
5,39c
BWF
−0,00016c
4,58a
4,49a
WBWF
−0,00677a
5,50d
4,87b
WOF
−0,00004d
5,12c
5,15c
MG
0,00022f
5,23c
5,30c
RF: rye flour, RG: rye grain, BF: barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
pH: rate of changes in pH values, pH0: initial pH, pHend: final pH.
a–gMeans within a column with different superscript letters are significantly different (P< 0.05).Table 8
Parameters of lactic acid concentration changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
acid [mg kg−1 h−1]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
−0,041c
276,52a
236,41a
RG
0,126g
404,90c
401,05d
BF
−0,122a
469,20e
361,41c
WBF
−0,060b
617,95f
603,81f
AF
3,752i
402,90c
2217,50i
AG
0,092f
883,81g
925,48g
BWF
−0,011d
395,75c
415,53d
WBWF
4,967j
416,12d
1122,91h
WOF
0,079e
255,74a
279,09b
MG
2,455h
359,89b
483,38e
RF: rye flour, RG: rye grain, BF:  barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of lactic acid concentration changes, c0: initial concentration of lactic acid, cend: final concentration of lactic acid.
a–iMeans within a column with different superscript letters are significantly different (P< 0.05).Table 9
Parameters of acetic acid concentration changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
acid [mg kg−1 h−1]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
0,016d
60,60b
63,15b
RG
0,069f
94,91d
117,07e
BF
0,007c
88,67d
78,48c
WBF
0,058e
94,44d
120,77e
AF
−0,091b
258,30f
227,02f
AG
0,098g
162,46e
225,97f
BWF
6,793h
0,05a
40,87a
WBWF
−0,236a
60,94b
37,78a
WOF
0,052e
92,55d
117,28e
MG
0,015d
82,33c
90,11d
RF: rye flour, RG: rye grain, BF:  barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of acetic acid concentration changes, c0: initial concentration of acetic acid, cend: final concentration of acetic acid.
a–hMeans within a column with different superscript letters are significantly different (P < 0.05).Table 10
Parameters of citric acid concentration changes during storage of fermented cereal and pseudocereal substrates.
Substrate
k
acid [mg kg−1 h−1]
c
0 [mg kg−1]
c
end [mg kg−1]
RF
−0,014d
170,02b
155,95a
RG
—
—
—
BF
−0,066f
216,76c
165,06a
WBF
−0,028e
478,37d
470,37d
AF
−0,732g
1121,94f
822,96f
AG
0,120b
515,66d
581,54e
BWF
0,003a
130,93a
136,86a
WBWF
−3,161h
557,95e
429,94c
WOF
—
—
—
MG
0,037c
228,06c
249,15b
RF: rye flour, RG: rye grain, BF:  barley flour, WBF: whole barley flour, AF: amaranth flour, AG: amaranth grain, BWF: buckwheat flour, WBWF: whole buckwheat flour, WOF: whole oat flour, MG: millet grain.
k
acid: rate of citric acid concentration changes, c0: initial concentration of citric acid, cend: final concentration of citric acid.
a–hMeans within a column with different superscript letters are significantly different (P< 0.05).


## 4. Conclusion

According to our results, we can say that probiotic strainLb. rhamnosus GG is able to grow and metabolize during the fermentation of cereal and pseudocereal substrates dealing to improve sensory quality of end products. We also found out that fermented cereal and pseudocereal substrates were stable during the 21-day storage period, except those prepared from amaranth flour, due to its rich nutritional composition. We are able to summarize that selected cereals and pseudocereals are suitable for developing new probiotic foods, which is in our interest.



---

*Source: 414303-2013-09-05.xml*