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Journal of the American College of Nutrition
ISSN: 0731-5724 (Print) 1541-1087 (Online) Journal homepage: https://www.tandfonline.com/loi/uacn20
Co-Culture Probiotic Fermentation of
Protein-Enriched Cereal Medium (Boza)
Sultan Arslan-Tontul & Mustafa Erbas
To cite this article: Sultan Arslan-Tontul & Mustafa Erbas (2020) Co-Culture Probiotic Fermentation of Protein-Enriched Cereal Medium (Boza), Journal of the American College of Nutrition, 39:1, 72-81, DOI: 10.1080/07315724.2019.1612796
To link to this article: https://doi.org/10.1080/07315724.2019.1612796
Published online: 13 May 2019.
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Co-Culture Probiotic Fermentation of Protein-Enriched Cereal Medium (Boza)
Sultan Arslan-Tontula and Mustafa Erbasb
a
Department of Food Engineering, Selcuk University, Agricultural Faculty, Konya, Turkey;bDepartment of Food Engineering, Akdeniz University, Engineering Faculty, Antalya, Turkey
ABSTRACT
Objective: Boza is a fermented cereal beverage which is produced by co-culture fermentation of lactic acid bacteria and yeasts. In addition to the nutritional properties of cereals used in the production, it is also suitable to be gaining functional properties by fermenting with probiotic microorganisms.
Methods: In this study, protein content of probiotic boza was increased by the addition of gluten, zein and chickpea flour and the volatile compounds formed during co-culture fermentation of the cereal medium with Lactobacillus acidophilus, Bifidobacterium bifidum and Saccharomyces boulardii were determined.
Results: It was determined that chickpea added boza provided the highest cell counts of Lactobacillus acidophilus (7.92 logs CFU/g), Bifidobacterium bifidum (7.32 log CFU/g) and Saccharomyces boulardii (3.26 log CFU/g) during storage. With the addition of gluten, the protein content of the sample was enriched four times more when compared with control boza. During fermentation and storage, a total of 36 different compounds were identified with the major compounds as 9,12-octadecadienoic acid, 9-octadecenoic acid, hexadecanoic acid and hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester. The concentration of volatile compounds generally decreased during storage of samples. According to Principle Cluster Analysis results, enriched protein samples had similar projections due to their fatty acid contents and the main difference was shown in the control sample.
Conclusions: The results of this study indicate that chickpea, single or mixture with cereals, can be a good substrate for probiotic microorganism production for acceptance as probiotic foods.
ARTICLE HISTORY
Received 1 November 2018 Accepted 25 April 2019
KEYWORDS
Legume; probiotic; functional foods; cereal fermentation; vola-tile compounds
Introduction
Boza is a traditional fermented cereal beverage mostly con-sumed in Turkey and the Balkans. Cereals such as millet, maize, oat, wheat, and rice or their mixture can be used in boza production (1). From this point of view, it is very suit-able to be fermented by probiotic lactic acid bacteria (LAB) which has a beneficial effect on human health when con-sumed in adequate amounts (106 CFU/g).
Cereal fermentation is a complex process, and several biochemical reactions occur in raw materials due to the enzyme activity of microorganisms (2). During fermentation, new compounds are formed such as organic acids, volatile compounds, ethanol, and aldehydes. However, there are lim-ited studies on the volatile compounds produced during cer-eal fermentation by probiotic microorganisms, and in most of the studies, malt and barley were used as raw materials (3–5). However, no study was conducted to determine the volatile compound composition of boza.
Cereal has been already used as a substrate for producing novel functional foods due to its prebiotic properties (6,7). Furthermore, previous research showed that cereals could also be used as a food carrier for the proliferation of pro-biotic LAB (8). However, their flour is generally used as raw material in fermentation as whole grains are not preferred. It is a fact that the total protein content of whole grain is
higher than that its flour. Aprodu and Banu (9) determined that the protein content of wheat, rye, triticale, hulled barley, and oat were higher in whole grain than their flour. Giacintucci et al. (10) reported that the protein content of emmer wheat whole grain and its flour were 14.4% and 11.8%, respectively. It was reported that valuable compo-nents such as proteins, fats, vitamins, and minerals had been found more abundantly in the outer layer of grains than in the inner layers of cereals (11,12). In addition, Yegin and Uren (13) found that the protein content of nine different boza samples varied between 0.51% to 0.99%.
None of the research in literature aimed to enrich boza with different protein sources and deeply investigated vola-tile compounds. Therefore, this study aimed to increase the protein content of probiotic boza and determine the volatile compounds formed during co-culture fermentation of a cer-eal medium by Lactobacillus acidophilus, Bifidobacterium bifidum, and Saccharomyces boulardii.
Materials and methods Materials
Maize semolina, wheat semolina, and sugar were purchased from a local market and used as raw materials. Gluten, zein,
CONTACTSultan Arslan-Tontul sultan.arslan@selcuk.edu.tr Selcuk University, Agricultural Faculty, Department of Food Engineering, 42130, Konya, Turkey. Color versions of one or more of the figures in the article can be found online atwww.tandfonline.com/uacn.
ß 2019 American College of Nutrition
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 2020, VOL. 39, NO. 1, 72–81
and chickpea flour were used for protein enrichment of boza. Gluten and zein were obtained from Zag Chem (Istanbul, Turkey). For chickpea flour production, whole grains were soaked in water for 8 hours and cooked for 1 hour. After cooking, it was dried at 60C overnight, grinded with a hammer mill (IKA, Werke, Germany), and sieved (500m, IKA, Werke, Germany).
Certified probiotic starter cultures, Lactobacillus acidoph-ilus LA-5 and Bifidobacterium bifidum BB-12, and Saccharomyces boulardii were obtained from Chr. Hansen (Denmark) and Reflor (Cedex, France), respectively. Chemical agents used in the analysis were obtained from Merck (Darmstadt, Germany) and Sigma (Taufkirchen, Germany).
Preparation of starter cultures
L acidophilus and B bifidum probiotic cultures were incu-bated in MRS broth (De Man, Rogosa and Sharpe Agar; Merck, Darmstadt, Germany) at 37C for 24 hours in anaer-obic conditions. For the growth of B bifidum, MRS broth was enriched with 0.05% L-cysteine. After incubation, cul-tures were harvested by centrifugation (14,000 rpm) at þ4C for 5 minutes and washed twice with sterile Ringer
solution (Merck, Darmstadt, Germany) for removal of broth residue. L acidophilus and B bifidum cell pellets were sus-pended in 0.9% sterilized saline solution, that is, 109 CFU/ mL. S boulardii culture was used in boza in their lyophilized form diluted to 105 CFU/mL in sterile water.
Production of probiotic boza
Boza was produced according to Arslan, Durak, Erbas, Tanriverdi, and Gulcan (1) with some modifications. A cereal mixture was prepared using maize and wheat semolina in a 3:1 ratio, respectively. Gluten, zein, and chickpea flour were added separately as 10% (w/w) to this cereal mixture (cereal mixture is in the flour form) to increase the protein content of the product. After preparation of protein-enriched raw cereal material, they were cooked with continuous stirring in boiling water for 90 minutes. The porridge of cooked cereals was cooled by adding water and homogenized for 20 minutes. After homogenization, sugar was added as a 10% of slurry and mixed. Each of suspended L acidophilus, B bifidum, and S boulardii probiotic cultures were added in the ratio of 1% (v/w) in boza samples. Boza samples containing three differ-ent protein sources were produced separately.
A control boza sample was produced with the same cook-ing and homogenization steps without the addition of pro-tein. Probiotic cultures and fermentation were carried out according to backslope fermentation. For this purpose, 5% natural inoculum obtained from a local market was added into the slurry.
Samples were fermented at 37C for 24 hours in a sample jar with a screw cap. At the end of the boza production pro-cess, four different formulations were obtained thus: control, gluten added, zein added, and chickpea flour added. After the fermentation period, they were stored atþ4C for 12 days in screw-cap glass jars. Samples (100 g) were taken over a
24-hour fermentation period (0, 12, and 24 24-hours) at 37C and over a 12-day storage period (0, 9, and 12 days) at 4C.
Enumeration of S boulardii
The cell population of S boulardii in the samples was enum-erated on spread plates of yeast extract glucose chloram-phenicol agar (YGC; Merck, Darmstadt, Germany) after suspending the sample (10 g) in 90 mL sterile Ringer solu-tion and making serial dilusolu-tions. The plates were incubated at 37C for 72 hours under aerobic conditions (1).
Enumeration of L acidophilus
L acidophilus was enumerated on Pour plates of MRS-BC agar with the pH adjusted to 6.2 (1 N HCl). Bromocresol green (0.2%; Merck, Darmstadt, Germany) was sterilized at 121C for 15 minutes and added to the agar medium to reach a final concentration of 2%. Clindamycin (50 mg/L; Sigma, Munich, Germany) was also added at 2 mL/L after filtration through a sterile 0.45mm membrane in agar medium. The plates were incubated at 37C for 72 hours in an anaerobic jar (1).
Enumeration of B bifidum
B bifidum was enumerated on Pour plates of TOS-MUP agar. TOS (transgalactosilated oligosaccharide; Sigma, Munich, Germany) agar was sterilized at 121C for 15 minutes and cooled to 55C, and lithium mupirocin solu-tion (50 mg/L; Sigma, Munich, Germany) was then added. Serial dilutions were made, and appropriate dilutions were poured unto plates for B bifidum enumeration. The plates were incubated at 37C for 72 hours an anaerobic jar (14).
Determination of pH and total titrable acidity
The pH value was determined using a pH meter (3410, WTW, Wellheim, Germany). For this purpose, a 5 g sample was diluted in 20 mL distilled water. This dilute was also used for determination of total titrable acidity. It was deter-mined by potentiometric titration with 0.1 N NaOH up to pH 8.1 while mixing continually on a magnetic stirrer after the pH measurement. The results were calculated from the following equation and given in terms of lactic acid (1).
% acid ¼ ml of NaOH used½ x 0:1 N NaOH½ x milliequivalent factor½ x 100½ Þ=grams of sample
Determination of total protein content
The total protein content of boza was determined by the Kjeldahl method and multiplied by a factor of 6.25 to deter-mine the crude protein content (15).
Determination of volatile compounds (VOCs)
VOCs in boza, formed during fermentation and storage, were determined according to Sereshti, Heidari, and Samadi (16) with some modifications. For the extraction of VOCs, a 2.5 g sample was weighed into a conical tube and 50 mL mixed solvent of methanol: Acetonitrile (38%:62%) was added. The tube was held in an ultrasonic bath (DL102H, Bandelin, Berlin Germany) for 22 minutes and centrifuged at 4000 rpm for 5 minutes to remove the insoluble parts of boza from the solution. Then, 1.3 mL chloroform containing 200mg/mL 1-octanol (as internal standard; Merck, Darmstadt, Germany) were added to the extract and ultraso-nicated for 2 minutes. After sonication, 2.5 mL solution was enriched with 10 mL 10% NaCl solution and centrifuged again at 4000 rpm for 5 minutes. Finally, lower chloroform phase was taken into a micro-vial, and 1mL sample was injected using the 1:15 split mode to the gas chromatog-raphy system with an HP-5MS column (30 m 0.25 mm 0.25 mm, Wilmington, DE, USA). The oven temperature was started at 40C and held for 1 minute, then increased to 250C at a rate of 5C/min and held for 20 minutes. The temperatures of the injection port and interface were set to 220C and 250C, respectively. Helium at a flow rate of 1 mL/min was used as the carrier gas.
The peaks were identified with mass spectral libraries of Wiley and NIST and Kovats retention index calculated from the retention times of alkane standard (Sigma, Munich, Germany). In addition, quantification of volatile compounds was performed through the use of internal standard (1-octa-nol) and analytes.
Statistical analysis
In this research, all boza productions and analyses were duplicated. All statistical calculations were performed using SAS Statistical Software (SAS Institute Inc., Cary, NC, USA). Values are presented as mean ± standard error. Significance was evaluated using analysis of variance followed by Duncan’s multiple range test (p < 0.05). In addition, the volatile compounds were also evaluated using principal com-ponent analysis (PCA) on the XLSTAT software (Addinsoft, New York, NY).
Results and discussion The viability of S boulardii
It was determined that protein type and fermentation time had a significant (p< 0.05) effect on S boulardii (Table 1). Yeast count of protein-added samples were similar but sig-nificantly higher than control produced with back slope fer-mentation culture. In addition, the incubation of boza was carried out at 37C which may be higher for other yeast strains found in the natural inoculum. Enujiugha and Badejo (17) reported Saccharomyces cerevisiae, Candida tropicalis, Candida glabrata, Geotrichum penicillatum, and Geotrichum candidum as yeast isolated from boza. Moreover, yeast count increased from 2.21 to 2.98 log CFU/ g in 24-hour fermentation.
There was a significant (p< 0.05) difference in S boulardii count in boza samples during storage mainly between con-trol and protein-added samples. The S boulardii count of protein-added samples were statistically similar during stor-age. However, the sample to which chickpea flour was added had a slightly higher S boulardii count (3.26 log CFU/g) than gluten- (2.99 log CFU/g) and zein- (2.89 log CFU/g) added samples. It was shown that the yeast viability remained constant during storage. This result could be as a result of low-temperature application in storage.
In this study, S boulardii was used as a probiotic starter culture by combining B bifidum and L acidophilus. There was limited research on the starter culture potentiality of S boulardii (18). Karaolis, Botsaris, Pantelides, and Tsaltas (19) studied the application of S boulardii in goat yogurt and found that S boulardii promoted the growth of other starter cultures, and also its cell population was 105–106 CFU/g during a 28-day storage period. Niamah (20) added different ratios (1%, 2%, and 3%) of S boulardii in yogurt made from whole cow’s milk and it was determined that the S boulardii count was changed 5.78 to 6.31 CFU/g after 21 days.
The viability of L acidophilus
It was indicated that protein type had an insignificant (p> 0.05) effect on L acidophilus counts while fermentation time significantly affected (p< 0.01) its growth (Table 1). During fermentation, average L acidophilus count was deter-mined to be 7.38 log CFU/g. This could be that the back-slope fermentation culture, used in the production of the control sample, contained L acidophilus or its similar strain.
Table 1. Probiotic microorganism counts (log CFU/g) of boza samples. Fermentation
Typea S boulardii L acidophilus B bifidum Gluten 2.97a± 0.18 7.29a± 0.34 6.11b± 0.14 Zein 2.81a± 0.18 7.36a± 0.18 6.95a± 1.05 Chickpea flour 2.95a± 0.13 7.56a± 0.37 7.16a± 0.23 Control 1.91b± 0.18 7.29a± 0.34 0.00c± 0.00 Significance — Time (hour)b 0 2.21b± 0.18 6.42b± 0.13 4.83b± 1.05 12 2.79a± 0.21 7.91a± 0.07 5.29a± 1.17 24 2.98a± 0.14 7.79a± 0.08 5.04ba± 1.13 Significance Storage
Typec S boulardii L acidophilus B bifidum Gluten 2.99ba± 0.17 7.29cb± 0.22 5.33c± 0.17 Zein 2.89ba± 0.17 7.03c± 0.19 6.41b± 0.12 Chickpea flour 3.26a± 0.08 7.92a± 0.07 7.32a± 0.38 Control 2.76b± 0.09 7.39b± 0.20 0.00d± 0.00 Significance Time (day)d 0 2.98a± 0.14 7.79a± 0.08 5.04a± 1.13 9 3.02a± 0.09 7.46b± 0.17 4.92a± 1.12 12 2.92a± 0.16 6.96c± 0.18 4.33b± 1.00 Significance —
Superscript letters beside the mean values in the same column note that are significantly different by Duncan’s multiple range test (p < 0.05). N: 6 for protein source and N: 8 for time.
aAverage of total 24 hours’ fermentation period belonging to each
boza sample.
bAverages of boza samples belonging to each sampling time at fermentation. c
Average of total 12 days’ storage period belonging to each boza sample.
dAverages of boza samples belonging to each sampling time at storage.
L acidophilus counts increased from 6.42 to 7.79 log CFU/g during fermentation time.
From the statistical results, it could be seen that protein type and storage time significantly (p< 0.05) affected L acid-ophilus counts. It is obvious that the main effect of protein source was observed during storage. The highest L acidoph-ilus count (7.92 log CFU/g) was determined in chickpea flour–added sample. This result may arise from bacterial growth supporting the effect of chickpea flour due to its micronutrient contents. It is known that microcomponents such as iron and manganese are essential nutrients for all microorganisms and could promote viability and cell growth (21). Also, Lactobacillus needs fermentable carbohydrates such as glucose, amino acids, water-soluble vitamins, nucleic acids, and minerals for growth (17). The sugar content of chickpea flour may support L acidophilus growth. It was determined that when glucose was added into the medium, the resistance of L acidophilus increased (22). Rizzello, Calasso, Campanella, De Angelis, and Gobbetti (23) noted that wheat–legume sourdough showed a higher viable num-ber of LAB compared to other sourdough bread samples. Kockova and Valik (24) researched the possibilities of using cereals, pseudo-cereals, and legumes as a substrate for growth of probiotic bacteria. Similarly, with our findings, they determined that an oat–chickpea mixture ensured high cell counts of Lactobacillus rhamnosus (7.58 log CFU/g) during fermentation (24).
L acidophilus viability decreased from 7.79 to 6.96 log CFU/g during storage. Low temperature, increased acidity, and competition for nutrients with other probiotic microor-ganisms might cause this decrease. It was reported in a pre-vious study that probiotic Bifidobacterium and Lactobacillus have a capability of binding micro elements in the medium (21).
The viability of B bifidum
It was indicated that protein type and time had a significant (p< 0.05) effect on B bifidum count during fermentation and storage time (Table 1).
During fermentation, the highest B bifidum count was obtained in samples containing chickpea flour– and zein-added samples as 7.16 and 6.95 log CFU/g, respectively. From these results, it can be deduced that chickpea, a leg-ume seed, may support the proliferation of probiotic B bifi-dum. Similar to these results, Agil et al. (25) asserted that lentils might have an increasing selective effect on probiotic L acidophilus and Bifidobacterium lactis microorganisms. However, no B bifidum count was detected in the control sample because the backslope fermentation culture used might not have contained B bifidum. In addition, B bifidum count increased from 4.83 to 5.04 log CFU/g during 24-hour fermentation. However, the final count of B bifidum was lower than L acidophilus at the end of the fermentation process. It was reported that grain flours were not suitable for the growth of Bifidobacterium and the addition of milk into the fermentation medium provided required nutrients such as casein (26).
The highest B bifidum count (7.32 log CFU/g) was deter-mined in chickpea-added samples during storage. This result might be explained by the fact that the addition of chickpea flour also increased B bifidum count under refrigerated con-ditions. However, the cell population of B bifidum continu-ously decreased throughout the 12-day storage, and it was determined to be 4.33 log CFU/g at the end of storage. A possible explanation for this result may be the low pH level of the cereal medium. It is known that Bifidobacterium are less acid-tolerant than Lactobacillus and their optimum growth pH is 6.5 to 7.0 (17). In a previous study, the growth of four different Bifidobacterium strains in the malt hydrol-ysate was reported to be very limited when the pH value of the medium was at 5.0 (27).
As a conclusion, the main simulative effects of protein enrichment of cereal medium on probiotics have been observed in the storage period. For L acidophilus and B bifi-dum the highest viability was detected in chickpea-added samples. Although L acidophilus and B bifidum viability decreased, it was within acceptable range for the named product as probiotic. Probiotics must be in adequate number (106–107 CFU/g) and consumed on a regular basis to show their beneficial effects to the gastrointestinal system (5).
pH and total titrable acidity
It is indicated in the present study that protein type and fer-mentation time had a significant effect (p< 0.01) on titra-tion acidity in fermentatitra-tion and storage (Table 2). However, the pH value was affected (p< 0.01) only at fermentation.
Table 2. pH and titratable acidity (%) content of boza samples. Fermentation
Type pH Titrable acidity (%) Gluten 4.81a± 0.53 1.79b± 0.39 Zein 4.85a± 0.43 1.94ba± 0.42 Chickpea flour 4.88a± 0.57 2.18a± 0.51 Control 4.79a± 0.30 1.33c± 0.22 Significance — Time (hour) 0 6.28a± 0.14 0.62c± 0.02 12 4.14b± 0.05 2.24b± 0.16 24 4.08b± 0.07 2.58a± 0.20 Significance Storage
Type pH Titrable acidity (%) Gluten 4.00a± 0.05 2.48c± 0.15 Zein 3.97a± 0.10 3.26ba± 0.33 Chickpea flour 3.88a± 0.08 3.71a± 0.24 Control 3.99a± 0.13 2.86bc± 0.50 Significance — Time (day) 0 4.08a± 0.07 2.58b± 0.20 9 4.05a± 0.03 2.78b± 0.19 12 3.74b± 0.06 3.88a± 0.32 Significance
Superscript letters beside the mean values in the same column note that are significantly different by Duncan’s multiple range test (p < 0.05). N: 6 for protein source and N: 8 for time.
aAverage of total 24 hours’ fermentation period belonging to each
boza sample.
bAverages of boza samples belonging to each sampling time at fermentation. c
Average of total 12 days’ storage period belonging to each boza sample.
The average pH values of samples during fermentation and storage were 4.83 and 3.96, respectively. The highest total titrable acidity was determined in the chickpea flour–added samples during fermentation. Moreover, the pH values of samples decreased while total titratable acidity level was increased continuously during fermentation. This result showed that the main reason for pH decrease was a microbio-logical activity during fermentation, and this finding is sup-ported with L acidophilus and B bifidum counts (Table 1). L acidophilus is a homo-fermentative bacteria species, and it produces lactic acid as an end product from the glycolysis of carbohydrates (28).
The pH and total titrable acidity of samples were 3.74 and 3.88% at the end of storage, respectively. It has been reported that food formulations that have pH levels of about 3.5 to 4.5 are desired because they enhance the stability and benefits to probiotic microorganisms (29,30) and inhibit the growth of coliforms (31,32). On the contrary, high pH val-ues positively related with acceptance of beverages (33).
The results are compatible with other studies that were aimed to develop cereal-based probiotic foods (24,30,34,35). It was determined that pH decreased from 5.8 to 3.1 to 3.71 after 12 hours’ production of maize porridge fermented by selected strains of probiotic bacteria (34). Kockova and Valik (24) researched the development of new cereal, pseudo-cereal, and legume-based probiotic foods and deter-mined that the pH value of samples ranged from 4.52 to 5.79 during fermentation and 4.86 to 5.98 in storage; the
lowest pH was determined in oat–chickpea sample. Yahyaoui, Bouzaiene, Aouidi, Aydi, and Hamdi (36) deter-mined that the pH value of a fermented breakfast cereal product carried by L rhamnosus decreased from 4.3 to 3.4 during storage. In addition, it should be noted that fermen-tation carried out in co-culture is more efficient in substrate acidification (30).
Total protein content
It was determined that protein type had a significant (p< 0.05) effect on the total protein content of samples. The highest protein content was detected in the gluten-added sample as 7.99 g/100g, followed by the zein- (7.54 g/100g) and chickpea flour– (6.10 g/100g) added sample, respectively. The total protein content of control was about 2.15 g/100g. Therefore, the protein content of the control sample could have been increased up to four times.
During the boza production process, water-soluble pro-teins decreased when compared with those of flour samples (37). It was determined that the protein content of boza samples was about 3.05 g/100g, while protein contents of the raw materials varied from 18.43 to 22.33 g/100g (37).
Nowadays, an important interest of food scientists is leg-ume supplementation of fermented probiotic cereal-based products, since one-third of the world’s population experien-ces protein-energy malnutrition. However, lysine is the major amino acids lacking in cereals, and most of the
Table 3. Volatile compounds detected in fermentation and storage of probiotic boza samples.
No. Compound Retention time Retention index
1 2-Pentanon 2.75 716
2 Butanoic acid methyl ester 3.09 739 3 2-methyl-Butanoic acid methyl ester 3.82 789
4 Octane 4.02 800 5 2-Hexanol 4.18 808 6 2-methyl- Octane 4.29 813 7 3-methyl-Octane 5.40 864 8 Ethenyl-Benzene 5.58 870 9 Nonane 6.28 900 10 2-methyl- Nonane 7.96 961 11 4-methyl- Nonane 8.04 964 12 Decane 9.07 1000 13 5-methyl-Decane 10.75 1057 14 2-methyl-Decane 10.85 1060 15 Nonanoic acid 16.92 1202 16 Tridecane 17.40 1288 17 4,6-dimethyl-Dodecane 18.42 1326 18 n-Decanoic acid 19.51 1302 19 1-Dodecanol 22.57 1487 20 Pentadecan 22.86 1499 21 2,4-di-tert-butylphenol 23.18 1500 22 Dodecanoic acid 24.37 1562 23 Hexadecane 25.24 1600 24 2,6,10,14-Tetramethylpentadecane 27.73 1710 25 Tetradecanoic acid 28.83 1761 26 Octadecane 31.44 1887 27 Nonadecan 31.66 1898 28 Hexadecanoic acid 32.95 1964 29 Cis-9,12-Octadecadienoic acid 35.41 2083 30 9,12-Octadecadienoic acid 36.31 2145 31 9-Octadecenoic acid 36.37 2148 32 Ethyl linoleate 36.63 2163 33 Octadecanoic acid 36.74 2169 34 (Z)-9-Octadecenamide 39.84 2351 35 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 42.34 2507
36 Squalene 48.67 2826
Table 4. The change of volatile compound concentrations (m g/g) in boza samples during fermentation, expressed as means ± SD. Group No Control Gluten Zein Chickpea flour 0 hour 12 hour 24 hour 0 hour 12 hour 24 hour 0 hour 12 hour 24 hour 0 hour 12 hour 24 hour Ketone 1 0.0d ± 0.0 0.0d ± 0.0 0.0d ± 0.0 15.5a ± 0.2 4.0b ± 1.7 1.3c ± 0.1 0.0d ± 0.0 0.0d ± 0.0 0.0d ± 0.0 0.0d ± 0.0 0.0d ± 0.0 0.0d ± 0.0 Esters 2 14.3a ± 3.0 15.3a ± 1.1 16.1a ± 1.3 17.1a ± 0.2 17.9a ± 3.6 11.6a ± 0.8 17.2a ± 2.5 15.7a ± 1.2 17.9a ± 2.4 16.7a ± 2.5 17.3a ± 3.8 18.8a ± 0.1 3 5.7a ± 1.0 4.3a ± 0.3 4.3a ± 0.5 4.4a ± 0.1 4.8a ± 1.1 3.6a ± 0.2 4.7a ± 0.7 4.3a ± 0.5 4.9a ± 0.8 4.7a ± 0.9 5.3a ± 1.3 5.2a ± 0.1 32 29.4b ± 4.2 33.0b ± 1.9 30.2b ± 7.1 27.2b ± 0.9 22.5b ± 2.6 22.4b ± 7.8 202.1a ± 5.6 47.7b ± 1 4 48.3b ± 6.6 25.4b ± 2.7 34.0b ± 1 2 36.8b ± 1.1 35 22.5g ± 1 1 50.4f ± 1 9 67.6e ± 1 9 181.9a ± 1 2 25.9g ± 5.1 130.2c ± 1 2 45.6f ± 2 1 73.1e ± 2 1 132.8c ± 1.3 161.3b ± 2 5 104.4d ± 6 0 36.4f ± 1 4 Total 71.9b ± 5.28 103ba ± 1.73 118ba ±2.51 230ba ±4.20 71.1b ± 9.11 167.8ba ± 8.98 269.6a ± 7.49 140.8ba ± 10.47 203.9ba ± 2.63 208.1ba ± 11.78 161.0ba ± 27. 95 97.2ba ± 6.91 Alcohols 5 13.8a ± 7.1 5.1ba ± 1.6 5.5ba ± 1.6 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 5.6ba ± 1.4 5.5ba ± 1.0 6.2ba ± 1.4 5.4ba ± 1.5 6.4ba ± 2.5 7.5ba ± 0.5 19 0.0b ± 0.0 2.9a ± 0.0 3.1a ± 0.4 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 2.5a ± 0.5 2.7a ± 0.1 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 Total 13.8a ± 5.02 8.0bac ± 1.1 8.6ba ± 0.85 0.0c ± 0.0 0.0c ± 0.0 0.0c ± 0.0 5.6bac ± 0.9 8.0bac ± 0.3 8.9ba ± 0.9 5.4bc ± 1.6 6.4bac ± 1.8 7.5bac ± 0.3 Hydrocarbon 4 5.3a ± 2.2 8.6a ± 1.9 9.5a ± 1.9 10.9a ± 0.7 10.8a ± 3.7 8.4a ± 0.6 10.8a ± 3.3 7.6a ± 1.4 9.4a ± 1.7 11.5a ± 2.6 11.0a ± 4.8 12.0a ± 0.9 6 4.6ba ± 0.7 3.7ba ± 0.6 3.9ba ± 0.6 7.6a ± 0.1 6.6ba ± 2.3 2.0b ± 0.5 4.4ba ± 1.1 3.2ba ± 0.7 3.9ba ± 0.7 4.5ba ± 1.1 4.4ba ± 1.5 4.8ba ± 0.2 7 3.4a ± 0.4 3.1a ± 0.3 3.4a ± 0.6 4.1a ± 0.2 4.5a ± 1.3 3.5a ± 0.3 3.7a ± 0.9 3.0a ± 0.3 3.6a ± 0.07 3.6a ± 0.9 3.6a ± 1.1 3.8a ± 0.4 8 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 4.6a ± 2.8 3.9a ± 0.7 2.8a ± 0.2 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 9 6.7d ± 0.0 0.0f ± 0.0 0.0f ± 0.0 21.8b ± 1.0 23.7a ± 7.2 17.9c ± 0.7 2.8e ± 0.4 2.6e ± 0.2 2.8e ± 0.0 2.2e ± 0.1 3.3e ± 0.0 0.0f ± 0.0 10 4.1b ± 0.9 2.8c ± 0.3 3.1bc ± 0.4 7.8a ± 0.5 2.4c ± 1.0 4.8b ± 1.2 3.1bc ± 0.6 2.8c ± 0.4 3.1bc ± 0.5 3.3bc ± 0.8 3.2bc ± 0.7 3.7bc ± 0.1 11 5.5a ± 1.5 3.8a ± 0.2 4.1a ± 0.7 2.5a ± 0.12 3.3a ± 0.8 2.4a ± 1.8 4.3a ± 0.9 3.4a ± 0.6 4.1a ± 0.8 4.2a ± 1.0 4.2a ± 1.1 4.8a ± 0.1 12 5.9b ± 1.7 3.0cb ± 0.3 3.2cb ± 0.1 2.9c ± 0.4 3.2cb ± 0.8 22.9a ± 1.6 3.2cb ± 0.6 3.6cb ± 0.7 2.9c ± 0.2 3.6cb ± 0.7 3.3cb ± 1.1 3.7cb ± 0.2 13 0.0d ± 0.0 0.0d ± 0.0 0.0d ± 0.0 9.0a ± 0.0 5.1b ± 0.2 2.5cb ± 1.8 0.0d ± 0.0 2.1cbd ± 0.0 2.6cb ± 0.0 2.3cbd ± 0.1 2.4cbd ± 0.0 2.6c ± 0.0 14 4.7a ± 0.0 2.3bac ± 0.2 2.6bac ± 0.1 0.0c ± 0.0 2.7bac ± 0.0 2.9bac ± 0.1 2.9bac ± 0.1 3.2bac ± 0.0 3.3bac ± 0.1 3.1bac ± 0.1 2.4bac ± 0.4 4.3ba ± 2.5 16 2.7a ± 0.1 2.6a ± 1.4 0.0b ± 0.0 0.0b ± 0.0 2.5a ± 0.9 3.4a ± 1.3 0.0b ± 0.0 2.6a ± 0.1 2.4a ± 0.0 2.6a ± 0.0 2.2a ± 0.0 0.0b ± 0.0 17 5.1ba ± 1.1 3.8bc ± 1.4 3.6bc ± 0.6 5.7a ± 0.5 4.6ba ± 0.9 2.7c ± 0.6 3.2bc ± 0.4 5.0ba ± 0.8 3.5bc ± 0.6 4.2ba ± 0.8 3.4bc ± 0.1 4.7ba ± 0.5 20 3.3ba ± 0.2 3.1ba ± 1.3 3.5ba ± 1.5 0.0c ± 0.0 3.4ba ± 0.8 5.0a ± 1.4 2.4b ± 0.1 3.9ba ± 0.6 3.5ba ± 3.1 3.5ba ± 0.9 3.0ba ± 0.6 4.2ba ± 0.1 23 0.0e ± 0.0 3.3cd ± 5.1 2.9d ± 0.0 3.7cd ± 0.05 7.5a ± 0.5 5.5b ± 1.6 3.5cd ± 0.2 4.8cb ± 1.8 3.5cd ± 0.6 4.2cb ± 1.8 4.8cb ± 2.0 3.5cd ± 0.0 24 4.3ed ± 0.5 6.4cb ± 1.3 5.7cd ± 0.1 9.8a ± 1.0 7.1b ± 2.6 3.8e ± 1.8 5.2cd ± 0.5 8.0b ± 1.6 4.4ed ± 0.6 7.1b ± 1.3 6.1cb ± 1.0 5.9cd ± 0.1 26 0.0b ± 0.0 3.2a ± 0.0 3.7a ± 0.5 2.4a ± 0.9 3.4a ± 1.2 3.1a ± 1.1 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 2.5a ± 0.0 2.3a ± 0.1 0.0b ± 0.0 27 2.8a ± 0.0 2.0a ± 1.6 3.2a ± 0.1 0.0b ± 0.0 2.9a ± 1.2 3.8a ± 0.7 0.0b ± 0.0 3.5a ± 1.2 3.2b ± 0.2 2.7a ± 0.1 2.1a ± 0.5 2.6a ± 0.2 Total 58.4a ± 0.70 51.7a ± 1.25 52.4a ± 0.55 92.8a ± 0.71 97.6a ± 1.77 97.4a ± 0.59 49.5a ± 0.80 59.3a ± 0.58 56.2a ± 0.79 65.1a ± 0.72 61.7a ± 1.17 60.6a ± 0.61 Acids 15 0.0b ± 0.0 0.0b ± 0.0 2.7ba ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 9.3a ± 0.1 10.8a ± 0.0 0.0b ± 0.0 18 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 7.5a ± 0.0 7.6a ± 0.0 0.0b ± 0.0 22 0.0e ± 0.0 3.5bc ± 1.3 2.9dc ± 3.5 1.6d ± 0.07 4.0bac ± 0.1 4.3bc ± 0.1 2.5dc ± 0.0 3.6bc ± 1.3 4.5ba ± 0.9 4.1ba ± 2.9 4.8ba ± 2.3 5.4a ± 1.6 25 3.2l ± 0.4 6.5g ± 1.2 5.4j ± 2.2 10.9d ± 0.6 11.3c ± 6.3 5.9 ı±0.6 3.6k ± 0.4 8.3e ± 0.6 7.8f ± 2.6 12.1a ± 2.2 11.9b ± 0.1 6.0h ± 0.8 28 168.9b ± 3 4 265.3ba ± 2 9 270.7ba ± 4 2 110b ± 0.4 301.5ba ± 1 2 239.3ba ± 5 5 268ba ± 3 2 331.9a ± 9 4 358.4a ± 5 1 291.5ba ± 8 7 298.4ba ± 8 7 301.1ba ± 1 1 29 3.8b ± 1.1 5.7a ± 0.9 5.3a ± 0.7 4.2ba ± 1.9 3.7b ± 0.4 4.0ba ± 1.4 3.8b ± 0.6 5.8a ± 1.3 6.2a ± 0.7 4.8ba ± 0.4 5.4a ± 0.6 6.6a ± 0.3 30 922.6a ± 7 8 1000a ± 8 0 1080a ± 291 678.7b ± 1 8 622.8b ± 104 809.1a ± 2 0 1128a ± 151 747.9a ± 166 938.6a ± 157 706.1a ± 4 7 915a ± 148 719.3a ± 2.2 31 173.1a ± 4 2 116.7ef ± 1 9 131.7ed ± 1 2 111.8f ± 6 3 112.8f ± 1 4 128.7e ± 1 9 167.1ba ± 2 7 155.9bc ± 8.9 173.5a ± 1 7 156.6bac ± 1 7 168.2ba ± 1 7 146.7dc ± 2.4 33 84.2b ± 3.9 47.1d ± 1 5 39.4ed ± 21.3 42.6d ± 1.1 36.2ed ± 4.0 27.8e ± 1.2 48.1d ± 1 3 68.7c ± 3 6 43.5d ± 10.5 113.0a ± 1 3 84.0b ± 3 2 62.9c ± 1 0 Total 1355.8a ± 27.8 1444.8a ± 2 6 1538.2a ± 9 5 959.8a ± 2 1 1092.3a ± 3 3 1219.1a ± 1 8 1621.1a ± 5 1 1322.1a ± 6 0 1532.5a ± 5 4 1305.1a ± 2 9 1506.1a ± 5 2 1248.2a ± 4.3 Phenols 21 3.8ed ± 0.9 10.2b ± 2.7 7.3c ± 0.9 11.1b ± 0.5 15.1a ± 4.1 4.5d ± 1.4 2.5e ± 0.0 11.2b ± 6.5 10.8b ± 0.9 6.2c ± 3.4 6.1c ± 4.0 9.4b ± 0.9 Miscallenous 34 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 2.7a ± 0.0 2.3a ± 0.0 0.0b ± 0.0 36 6.7d ± 2.1 6.4d ± 0.7 11.1b ± 0.7 8.3cd ± 0.6 6.8d ± 1.0 14.2a ± 0.3 10.7b ± 0.9 9.6cb ± 2.7 10.3b ± 1.0 10.2b ± 1.1 13.6a ± 0.8 10.6b ± 0.4 Total 6.7a ± 1.5 6.4a ± 0.5 11.1a ± 0.5 8.3a ± 0.4 6.8a ± 0.7 14.3a ± 0.2 10.7a ± 0.6 9.6a ± 1.9 10.3a ± 0.7 12.9a ± 0.8 15.9a ± 0.5 10.6a ± 0.3 Superscript letters beside the mean values in the same column note that are significantly different by Duncan ’s multiple range test (p < 0.05).
Table 5. The change of volatile compound concentrations (m g/g) in boza samples during storage, expressed as means ± SD. Group No Control Gluten Zein Chickpea flour 0 day 3 day 12 day 0 day 3 day 12 day 0 day 3 day 12 day 0 day 3 day 12 day Ketone 1 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 1.3a ± 0.1 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 Ester 2 16.1ba ± 1.3 15.9ba ± 1.8 16.8ba ± 1.2 11.6c ± 0.8 15.9ba ± 1.0 15.7ba ± 3.6 17.9ba ± 2.4 17.1ba ± 2.7 15.9ba ± 2.8 18.8a ± 0.1 16.8ba ± 2.3 15.5ba ± 2.1 3 4.3a ± 0.5 4.3a ± 0.5 4.5a ± 0.2 3.6a ± 0.2 4.2a ± 0.5 4.3a ± 1.2 4.9a ± 0.8 4.7a ± 0.9 4.3a ± 0.8 5.2a ± 0.1 4.4a ± 0.7 4.3a ± 0.8 32 30.2ba ± 7.1 31.1ba ± 7.9 34.1ba ± 1 1 22.4b ± 7.8 25.2b ± 5.4 31.2b ± 1.5 48.3ba ± 6.6 46.8ba ± 1 3 46.6ba ± 9.8 36.8ba ± 1.1 69.2ba ± 5.4 77.3a ± 2 9 35 67.6c ± 1 9 41.9d ± 2 2 0.0e ± 0.0 130.2a ± 1 2 64.9c ± 0.0 63.2c ± 4.7 132.8a ± 1.3 83.8b ± 1 7 42.6d ± 0.0 36.4d ± 1 4 124.1a ± 1 9 0.0e ± 0.0 Total 118.2ba ± 8.5 93.2ba ± 9.8 55.4b ± 5.3 167.8ba ± 5.7 110.2ba ± 2.5 114.4ba ± 1.7 203.9ba ± 2.6 152.4ba ± 7.8 109.4ba ± 4.4 97.2ba ± 6.8 214.5a ± 8.3 97.1ba ±1 4 Alcohols 5 5.5ba ± 1.6 5.5ba ± 1.4 4.6ba ± 0.5 0.0c ± 0.0 5.6ba ± 1.2 5.3ba ± 0.9 6.2b ± 1.4 5.9ba ± 1.4 3.4bc ± 0.4 7.5a ± 0.5 5.7ba ± 1.4 4.1ba ± 0.1 19 3.1ba ± 0.4 2.6ba ± 0.2 3.1ba ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 2.7ba ± 0.1 3.7a ± 0.0 2.8ba ± 1.7 0.0b ± 0.0 0.0b ± 0.0 2.7ba ± 0.0 Total 8.6a ± 0.8 8.1ba ± 0.8 7.7ba ± 0.3 0.0b ± 0.0 5.6ba ± 0.8 5.3ba ± 0.6 8.9a ± 0.9 9.6a ± 0.9 6.2ba ± 0.9 7.5ba ± 0.3 5.7ba ± 0.9 6.8ba ± 0.1 Hydrocarbon 4 9.5a ± 1.9 7.2a ± 1.3 9.8a ± 3.0 8.4a ± 0.6 8.4a ± 1.6 9.1a ± 0.7 9.4a ± 1.7 10.6a ± 3.8 7.0a ± 2.2 12.0a ± 0.9 7.1a ± 0.6 9.1a ± 2.6 6 3.9ba ± 0.6 3.6ba ± 0.6 4.1ba ± 1.2 2.0b ± 0.5 3.4baa ± 0.7 3.9ba ± 1.7 3.9ba ± 0.7 4.3ba ± 1.4 3.1ba ± 0.7 4.8a ± 0.2 3.2b ± 0.4 3.7ba ± 0.8 7 3.4ba ± 0.6 3.0ba ± 0.5 3.3ba ± 0.7 3.5ba ± 0.3 3.2ba ± 0.6 4.5ba ± 2.1 3.6ba ± 0.07 5.0a ± 0.7 2.6b ± 0.6 3.8ba ± 0.4 3.0ba ± 0.5 3.3ba ± 0.7 8 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 2.8a ± 0.2 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 9 0.0e ± 0.0 0.0e ± 0.0 0.0e ± 0.0 17.9a ± 0.7 13.3b ± 5.5 3.6c ± 1.5 2.8d ± 0.0 2.9d ± 0.4 0.0e ± 0.0 0.0e ± 0.0 0.0e ± 0.0 0.0e ± 0.0 10 3.1a ± 0.4 2.8a ± 0.3 3.2a ± 0.6 4.8a ± 1.2 3.3a ± 1.4 3.9a ± 1.8 3.1a ± 0.5 4.6a ± 0.5 2.5a ± 0.4 3.7a ± 0.1 2.7a ± 0.4 3.7a ± 0.2 11 4.1ba ± 0.7 3.8ba ± 0.7 4.0ba ± 1.0 2.4b ± 1.8 3.5ba ± 0.7 7.7a ± 5.0 4.1ba ± 0.8 4.2ba ± 1.2 3.3ba ± 0.4 4.8ba ± 0.1 3.5ba ± 4.5 3.9ba ± 0.9 12 3.2b ± 0.1 3.2b ± 0.5 3.1b ± 0.5 22.9a ± 1.6 3.7b ± 1.5 3.2b ± 0.3 2.9b ± 0.2 3.4b ± 0.6 3.0b ± 0.5 3.7b ± 0.2 2.8b ± 0.2 3.0b ± 0.5 13 0.0c ± 0.0 0.0c ± 0.0 2.8ba ± 0.0 2.5bc ± 1.8 2.9ba ± 1.2 3.1a ± 1.3 2.6bc ± 0.0 2.5bc ± 0.1 0.0c ± 0.0 2.6bc ± 0.0 0.0c ± 0.0 0.0c ± 0.0 14 2.6b ± 0.1 2.6b ± 1.3 2.9b ± 0.0 2.9b ± 0.1 2.7b ± 1.1 3.7ba ± 1.5 3.3ba ± 0.1 3.4ba ± 0.3 2.5b ± 0.1 4.3a ± 2.5 2.6b ± 0.0 2.9b ± 0.1 16 0.0b ± 0.0 1.3ba ± 1.2 2.3a ± 0.0 3.4a ± 1.3 2.7a ± 1.1 0.0b ± 0.0 2.4a ± 0.0 2.4a ± 0.0 2.4a ± 0.4 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 17 3.6b ± 0.6 4.4ba ± 1.2 5.7a ± 0.0 2.7b ± 0.6 5.2a ± 2.2 4.3ba ± 0.6 3.5b ± 0.6 3.3b ± 0.5 4.3ba ± 0.8 4.7ba ± 0.5 4.0ba ± 0.2 2.6b ± 1.1 20 3.5a ± 1.5 3.8a ± 1.1 3.6a ± 1.4 5.0a ± 1.4 4.3a ± 1.8 4.0a ± 0.6 3.5a ± 3.1 3.5a ± 0.4 4.0a ± 1.1 4.2a ± 0.1 3.3a ± 0.4 3.4a ± 0.8 23 2.9ba ± 0.0 2.8ba ± 0.1 3.6ba ± 0.3 5.5a ± 1.6 3.7ba ± 0.8 2.8ba ± 1.2 3.5ba ± 0.6 2.6b ± 0.3 2.9ba ± 0.9 3.5ba ± 0.0 2.9ba ± 0.1 3.0ba ± 0.4 24 5.7ba ± 0.1 6.2a ± 1.5 5.4ba ± 1.9 3.8c ± 1.8 5.9ba ± 1.4 6.1a ± 1.1 4.4bc ± 0.6 4.9bac ± 0.9 6.2a ± 1.4 5.9ba ± 0.1 6.1a ± 0.3 4.7bac ± 1.4 26 3.7a ± 0.5 3.7a ± 1.8 3.6a ± 0.0 3.1ba ± 1.1 0.0b ± 0.0 2.6ba ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 27 3.2a ± 0.1 1.8a ± 1.7 2.5a ± 0.1 3.8a ± 0.7 3.2a ± 1.3 3.6a ± 1.5 3.2a ± 0.2 2.8a ± 0.3 3.3a ± 0.6 2.6a ± 0.2 2.6a ± 0.0 3.7a ± 0.0 Total 52.4a ± 0.5 50.2a ± 0.6 59.9a ± 0.8 97.4a ± 7.4 69.4a ± 1.2 66.1a ± 1.2 49.2a ± 0.8 60.4a ± 0.9 47.1a ± 0.6 60.6a ± 0.6 43.8a ± 1.1 47.0a ±0.7 Acids 15 2.7a ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 0.0b ± 0.0 18 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 22 2.9b ± 3.5 4.4ba ± 1.1 3.8ba ± 0.9 4.3ba ± 0.1 3.5ba ± 0.5 4.0ba ± 1.8 4.5ba ± 0.9 2.8b ± 0.5 3.0ba ± 0.2 5.4a ± 1.6 2.8b ± 0.1 3.9ba ± 1.2 25 5.4b ± 2.2 15.2a ± 4.5 12.9ba ± 3.1 5.9b ± 0.6 8.3ba ± 3.0 10.3ba ± 0.6 7.8ba ± 2.6 9.2ba ± 3.5 11.4ba ± 4.4 6.0b ± 0.8 5.0b ± 0.8 13.ba3 ± 9.5 28 270.6bc ± 4 2 276.4bc ± 7 8 287.2bac ± 8 7 239.3 ± 5 5 246.7c ± 3 4 278.7bc ± 1 2 358.4ba ± 5 1 333.9ba ± 8 7 176.5c ± 4 9 301.1bac ± 1 1 256.6bc ± 3 3 382.8a ± 9 7 29 5.3a ± 0.7 4.7a ± 1.2 5.6a ± 1.4 4.0a ± 1.4 3.7a ± 0.7 4.0a ± 1.4 6.2a ± 0.7 5.6a ± 0.9 5.7a ± 1.1 6.6a ± 0.3 5.8a ± 1.4 7.7a ± 1.7 30 1080b ± 291 1295a ± 147 1089b ± 211 809.1cd ± 2 0 615.5e ± 7.7 573.2e ± 9 3 938.6cb ± 157 881.5cd ± 3 1 877.9cd ± 120 719.3ed ± 2.2 639.6e ± 160 935.2bc ± 253 31 131.7a ± 1 2 123.3a ± 2 4 131.6a ± 3 7 128.7a ± 1 9 135.7a ± 5 6 113.8a ± 3 2 173.5a ± 1 7 154.1a ± 5.3 103.4a ± 5.6 146.7a ± 2.4 107.8a ± 2 4 155.1a ± 2 3 33 39.4b ± 21.3 60.1a ± 1 7 61.2a ± 1 6 27.8b ± 1.2 53.0ba ± 1 5 62.1a ± 4.3 43.5ba ± 10.5 52.4ba ± 2 7 50.6ba ± 4.9 62.9a ± 1 0 25.1b ± 8.6 51.9ba ± 9.5 Total 1538a ± 9 4 1779a ± 5 0 1591a ± 7 0 1219a ± 1 8 1066a ± 1 9 1046a ± 3 0 1532a ± 5 1 1439a ± 2 8 1228a ± 4 0 1248a ± 4.2 1042a ± 5 2 1549a ± 8 4 Phenols 21 7.3a ± 0.9 8.6a ± 2.1 3.3a ± 0.9 4.5a ± 1.4 9.9a ± 0.5 3.8a ± 0.6 10.8a ± 0.9 2.9a ± 0.2 3.2a ± 0.7 9.4a ± 0.9 9.2a ± 6.8 12.2a ± 5.1 Miscallenous 34 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 36 11.1a ± 0.7 8.8a ± 1.8 8.0a ± 3.1 14.2a ± 0.3 9.6a ± 0.1 6.3a ± 1.5 10.3a ± 1.0 10.9a ± 2.1 9.8a ± 2.2 10.6a ± 0.4 9.8a ± 2.7 8.1a ± 1.2 Total 11.1a ± 0.5 8.8a ± 1.3 8.0a ± 2.2 14.2a ± 0.2 9.6a ± 0.1 6.3a ± 1.1 10.3a ± 0.7 10.9a ± 1.5 9.8a ± 1.5 10.6a ± 0.3 9.8a ± 1.9 8.1a ± 0.8 Superscript letters beside the mean values in the same column note that are significantly different by Duncan ’s multiple range test (P < 0.05).
world’s population consumes cereal-based products. Therefore, enhancement of cereal fermentation media by different protein sources such as legumes and their deter-mination of probiotic growth potentiality is an important research area (17).
VOCs
The VOCs detected in boza samples during fermentation and storage are shown in Table 3 and their concentrations (mg/g) in Table 4 and Table 5. During boza fermentation and storage a total of 36 VOCs were identified.
The main VOCs, detected during cereal fermentation, were reported to be formed as a result of carbohydrate and amino acid metabolisms (33). During fermentation, the most abundant compounds were 9,12-octadecadienoic acid, 9-octadecenoic acid, hexadecanoic acid, and hexadeca-noic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester. These findings are in agreement with Salmeron, Fucinos, Charalampopoulos, and Pandiella (3), who determined that the major VOCs were oleic and linoleic acid after fermenta-tion of cereals by Lactobacillus plantarum. Also, Ogunremi, Agrawal, and Sanni (38) determined 9,12-octadecadienoic acid methyl ester as the major aromatic compounds in a substrate mix fermented with a probiotic strain of Pichia kudriavzevii. In addition, esters were attributed to major aromatic and flavor compounds formed during LAB and yeast fermentation (39). 9,12-octadecadienoic acid and 9-octadecenoic acid can act as precursors for flavor com-pounds such as methyl ketones, alcohols, and lactones, but these acids could be attributed to yeast activity more than LAB due to their low lypolitic activities (33).
In previous studies, it was reported that cereal type could affect the volatile composition of the final product (40). Similarly, in this study, some compounds were detected as specific to the formulation. 2-pentanon and ethenylbenzene were formed only in the gluten-added boza sample while nonanoic acid, decanoic acid, and (Z)-9-octadecenamide were evaluated in chickpea flour–added boza samples. Also, 2-hexanol and nonane were determined in all samples to expect gluten-added boza samples. Moreover, some com-pounds were formed during late fermentation stages such as tridecane, nonadecane, and 1-dodecanol. The concentration of VOCs generally decreased during storage of boza samples. It was reported that the relative concentration of most VOCs formed in cereal medium decreased (3).
PCA is a useful technique for making a good inference among a large quantity of data. It has also successfully man-aged to reduce the dimensionality of data set down from 36 (compound size) 4 (boza sample) 3 (different fer-mentation time) 3 (storage time) to 4 clusters.
PCA projections of samples obtained during fermentation and storage are shown in Figure 1 and 2; respectively, the first two principal components showed 99% variation in the data. Generally, protein-enriched samples had similar PCA projections due to their acid VOCs. There is a noticeable difference between the values for acid VOCs which are roughly similar in gluten-, zein-, and chickpea-added boza samples, which is usually significantly higher or lower. In the fermentation stage, control and chickpea-added samples showed similarity with regard to acid VOCs. However, the composition and amounts of acid VOCs changed during the storage period, and the control was in a different cluster from gluten-, zein-, and chickpea-added boza. The production process and natural inoculum may have affected
the chemical composition of the starter used in the con-trol sample.
Conclusion
The results of this study indicate that plant protein may be used for protein enrichment of cereal-based fermented func-tional foods. Therefore, legumes such as chickpea, single or mixture with cereals, can be a good substrate for probiotic microorganism production for acceptance as probiotic foods. According to the results, L acidophilus was more stable in cereal media than B bifidum during fermentation and stor-age time. By the addition of gluten, the protein content of the boza sample could have been increased four times. In addition, during boza fermentation and storage, 36 VOCs were identified. The main VOCs determined during pro-biotic fermentation of cereal substrates were 9,12-octadeca-dienoic acid, 9-octadecenoic acid, hexadecanoic acid, and hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester.
ORCID
Sultan Arslan-Tontul http://orcid.org/0000-0003-1557-7948
Mustafa Erbas http://orcid.org/0000-0002-9485-2356
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