Multi-Cereal Beverage Fermented by Lactobacillus Helveticus and Saccharomyces Cerevisiae Jing Ai, Ai-Li Li, Ben-Xian Su, and Xiang-Chen Meng
A novel multi-cereal-based fermented beverage with suitable aroma, flavor, and pH fermented by lactic acid bacteria and Saccharomyces cerevisiae was developed. Twenty-seven lactobacilli strains were screened for acid production (pH and titratable acidity) in a mixture of malt, rice, and maize substrates. It was found that Lactobacillus helveticus KLDS1.9204 had the greatest acid production among 27 lactobacilli tested. The fermentation performance of L. helveticus KLDS1.9204 was also assayed and the fermentation parameters were optimized using Plackett–Burman design and steepest ascent method. L. helveticus KLDS1.9204 showed good proteolytic capability, however, the strain could not utilize starch. The optimum substrate consisted of 50% malt (25 g/100 mL), 25% rice (20 g/100 mL), and 25% maize (30 g/100 mL). The inoculum was 5% with a ratio of S. cerevisiae to L. helveticus KLDS1.9204 of 2.5:1. The optimum temperature was 37 °C and the time was 22 h. Lastly, the quality of the multi-cereal-based fermented beverage was evaluated. This beverage was light yellow, transparent, and it tasted well with a pleasant acid and a unique flavor of cereals. The beverage was rich in free amino acids and organic acids. The pH and titratable acidity of the beverage were 3.5 and 29.86 °T, respectively. The soluble solids content of the beverage was 6.5 °Brix, and the alcohol content was 0.67%.
Abstract:
A novel multi-cereal-based fermented beverage with suitable aroma, flavor, and pH fermented by lactic acid bacteria and yeast was developed. A combination of starters, including Saccharomyces cerevisiae and Lactobacillus helveticus KLDS1.9204, was suitable for fermentation of cereal. There is potential for production in large scale.
Practical Application:
Introduction There is a considerable amount of work done on cerealcontaining foods fermented by lactic acid bacteria or yeast or their combination. Some probiotic beverages were produced by fermentation of cereals, but most of which took single cereal substrates as delivery vehicles for potential probiotic lactic acid bacteria. Gupta and others (2010) have optimized the process for development of an oats beverage fermented by Lactobacillus plantarum. Coda and others (2012) manufactured functional emmer beverages fermented by L. plantarum 6E. Kedia and others (2007) have used mixed cultures (yeast and L. reuteri) for fermentation of cereal beverages. Rathore and others (2012) have also used mixed cereals including barley and malt as substrates for the production of beverage, and L. plantarum and L. acidophilus were used as starter cultures. So far, little work has been reported on the development of multi-cereal beverages fermented by mixed cultures. Cereals have complex nutrient composition and when they are mixed in certain proportions, the properties of the product can be considerably modified. The nutrient availability for microorganisms may change when different cereals are mixed, furthermore, the growth and metabolism of microorganisms in the mixture may change.
MS 20141087 Submitted 6/24/2014, Accepted 3/2/2015. Authors Jing Ai, Ai-Li Li, Ben-Xian Su, and Xiang-Chen Meng are with Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural Univ., Harbin 150030, China. Authors Jing Ai, Ai-Li Li, Ben-Xian Su, and Xiang-Chen Meng are with Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural Univ., Harbin 150030, China. Direct inquiries to author Meng (E-mail: [email protected]).
R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12859 Further reproduction without permission is prohibited
An appropriate selection of substrate composition and microbial strains is necessary to efficiently control the composition of the metabolic end-products. The main aim of this work is to develop a novel multi-cereal (malt, rice, and maize) beverage fermented by lactic acid bacteria and S. cerevisiae. Fermentation performance of the starter strains was also analyzed. Fermentation parameters including compositional cereal substrates (ratio of malt, rice, and maize), inoculum level, ratio of S. cerevisiae to L. helveticus KLDS1.9204, fermentation temperature and fermentation time were optimized. The pH, titratable acidity, soluble solids content (SSC), free amino acids, and organic acids of beverage were determined.
Materials and Methods Cereal fermentation substrates Malt, maize powder, and rice were purchased from local markets. The malt was milled using a FW100 high-speed smashing machine (Taisite Instruments, Tianjin, China) fitted with a 0.5 mm aperture sieve. Malt fermentation substrate was prepared by dissolving 25 g of malt powder in 100 mL of distilled water and the mixture was then kept in a water bath at 65 °C for 2.5 h. The mixture was then filtered using 8 layers of gauze and finally autoclaved at 121 °C for 15 min. Rice and maize fermentation substrates were prepared by dissolving 20 g of rice or 30 g of maize powder in 100 mL of distilled water. Rice and maize substrates were boiled for 20 min by a wire coil heater (Yongguangming Instruments, Beijing, China) and starches in the fermentation substrates were gelatinized. Starches were hydrolyzed by the addition of 0.5% Vol. 80, Nr. 6, 2015 r Journal of Food Science M1259
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Keywords: Cereal, fermented beverage, Lactobacillus helveticus, Saccharomyces cerevisiae
Multi-cereal fermented beverage . . . (w/w) of α-amylase (20000 μ/g, Zhaodong, China) in a water bath at 90 °C for 1 h. The substrates were cooled to 65 °C and 1% (w/w) of glucoamylase (100000 μ/g, Hunan, China) was added. The substrates were then transferred to a bath at 65 °C for 2 h. Finally, they were filtered and autoclaved at 121 °C for 15 min.
Microbiological propagation Twenty-seven lactobacilli strains obtained from Bacterial Collection (Key Laboratory of Dairy Science, Northeast Agricultural University, Harbin, China) including 17 strains of L. plantarum, 6 strains of L. paracasei, 2 strains of L. rhamnosus, 1 strain of L. helveticus, and 1 strain of L. brevis were propagated for 12 h at 37 °C in deMan Rogosa Sharp (MRS) broth (Nguyen and Loiseau 2007) from frozen stock cultures. Then, the starter culture was obtained by overnight incubation at 37 °C in MRS broth. Dried S. cerevisiae (Anqi yeast, Yi Chang, Hu Bei, China) was grown in 2% sucrose solution for 2 h at room temperature and used for inoculation immediately.
Table 1–Levels of the variables tested in Plackett–Burman design. Variable code X1 X2 X3 X4 X5 X6 ,X7
Variables Cereal substrates ratio of malt, rice and maize Inoculum amount (%) Cell ratio of S. cerevisiae and L. helveticus KLDS1.9204 Fermentation time (h) Fermentation temperature (°C) Dummy factors
Low Low level (−)
High High level (+)
6:3:1
5:3:2
4 2:1
9 4:1
16 25
22 37
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Composition of mixed cereal substrates and samples fermented by L. helveticus Mixed cereal substrates were obtained by mixing malt substrate, rice substrate, and maize substrate with the ratio of 5:2:3 (v/v). A 1% inoculum of L. helveticus KLDS1.9204 was added to the mixed cereal substrates and incubated for 12 h. Sampling was performed Screening of lactobacilli strains at the end of the fermentation. Some physical–chemical indices Cereal substrates were fermented by each of the 27 lactobacilli of the substrates and the fermented samples, including the pH, strains for 12 h at 37 °C with 1% inoculation. Sampling was done at titratable acidity, SSC, free amino acids, and total reducing sugars, 12 h; then, the pH and titratable acidity of the fermented product were analyzed. were determined. Fermentation characteristics of Lactobacillus helveticus KLDS1.9204 Growth curve. L. helveticus KLDS1.9204 was added to multicereal substrates with a 1% inoculation and incubated at 37 °C for 24 h. Sampling was performed every 2 h. Growth was monitored by determination of colony-forming units (CFU) in the fermented mixture as described by Rimaux and others (2012). Cell counts (CFU/mL) were obtained by preparing 10-fold serial dilutions of the samples in saline (0.9%, wt/vol NaCl solution) and plating appropriate dilutions onto MRS agar. After incubation at 37 °C for 24 h, CFU were enumerated. Protein degrading and carbohydrate hydrolyzing capabilities. Extracellular proteolytic activity and utilization of starch were assessed by using plate count agar with 50% skim milk and starch agar respectively (Blaiotta and others 2012). Overnight cultures in MRS (10 μL) for L. helveticus KLDS1.9204 were dropped onto the agar surfaces and incubated at 37 °C for 48 h. The diameters of the halo zone on the agar plate were then measured. The hydrolyzing capability of the tested strains was classified as positive when the diameters of clear zone were more than 1 mm. Each assay was performed in triplicate. Protease activity. Protease activity was measured using casein as substrate by Folin-phenol method (Beganovic and others 2013). The cell-free supernatant of L. helveticus KLDS1.9204 (1 mL) was added to 1 mL casein solution (2%, w/v in 0.1 mol/L phosphate buffer, pH 7.2) and the reaction mixture was incubated at 40 °C for 10 min. Then, 2 mL of trichloroacetic acid (TCA) solution (0.4 M TCA) was added for stopping the reaction. After incubation at 40 °C for 20 min, the reaction mixture was centrifuged and the TCA soluble peptides in the supernatant fraction were measured by the method of Todd (1949) with tyrosine (National Institute for Food and Drug Control, China) as the reference compound. One unit of protease activity was defined as the amount of enzyme required to release 1 μg of tyrosine per min. M1260 Journal of Food Science r Vol. 80, Nr. 6, 2015
Optimization of fermentation parameters using Plackett–Burman design and steepest ascent Determination of significant variables using PB design. The Plackett–Burman (PB) design (Naveena and others 2005) for 7 variables included: cereal substrates ratio of malt, rice and maize, inoculum amount, cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, fermentation time, fermentation temperature, and two dummy factors at two levels (+1 and −1) (Table 1) was used for screening fermentation parameters that significantly influenced titratable acidity and alcohol production. The levels of these factors were determined in one-factor experiment (data not shown). In the current study, a 12-run PB design was applied to evaluate 7 factors (including 2 dummy variables). All experiments were carried out in triplicate and the averages of titratable acidity and alcohol production were taken as response. In the experimental design, each row represents an experiment and each column represents an independent variable (Table 2). The signs + and – represent the two different levels (high and low) of the independent variable under investigation. Steepest ascent. Steepest ascent (SA) design (Zhu and others 2012) was used to move rapidly toward the neighborhood of the optimum response. The zero level of PB design was identified as the base point of SA path. The step along the path was determined by the estimated coefficient and practical experience. Experiments were performed along the SA path until the response showed no further increase. Table 3 shows the SA used in this study. The experiments were performed to determine a suitable direction by increasing or decreasing the concentrations of variables according to the results of PB design. Evaluation of multi-cereal fermented beverage Malt, rice, and maize substrates were mixed with the ratio obtained in the SA. Then, L. helveticus KLDS1.9204 and S. cerevisiae were inoculated in the mixed cereal substrates and incubated under the optimum fermentation conditions according to the results obtained in the SA. Fermentations were carried out at 37 °C in
Multi-cereal fermented beverage . . . Table 2–The Plackett–Burman design for 7 variables coded values with titratable acidity and alcohol production as response. Run
X1
X2
X3
X4
X5
X6
X7
Titratable acidity (°T)
Alcohol production (%)
1 2 3 4 5 6 7 8 9 10 11 12
1 1 −1 1 −1 −1 −1 1 −1 1 −1 1
1 −1 −1 1 1 1 1 1 −1 −1 −1 −1
−1 1 1 1 1 −1 1 −1 −1 −1 −1 1
1 −1 1 −1 −1 −1 1 1 −1 −1 1 1
−1 −1 1 1 1 −1 −1 1 −1 1 1 −1
−1 −1 −1 1 −1 1 1 −1 −1 1 1 1
−1 1 1 −1 −1 1 1 1 −1 1 −1 −1
29.82 ± 1.11 29.57 ± 1.79 26.60 ± 1.67 28.48 ± 2.96 25.43 ± 1.18 25.63 ± 4.00 25.02 ± 3.69 28.27 ± 1.63 27.47 ± 1.89 31.58 ± 1.02 28.02 ± 0.99 30.00 ± 1.68
0.67 ± 0.16 0.83 ± 0.48 1.00 ± 0.13 0.30 ± 0.11 0.63 ± 0.15 0.70 ± 0.28 0.70 ± 0.49 0.37 ± 0.08 1.10 ± 0.33 1.00 ± 0.44 1.60 ± 0.22 0.97 ± 0.08
X1 : Cereal substrates ratio of malt, rice and maize. X2 : Inoculum amount (%). X3 : Cell ratio of S. cerevisiae and L. helveticus KLDS1.9204. X4 : Fermentation time (h) X5 : Fermentation temperature (°C) X6 , X7 : Dummy factors.
Table 3–Effects of the variables and statistical analysis of the PB design.
Variables X1 X2 X3 X4 X5 X6 X7
Effect 3.2583 −1.7639 −0.9472 −0.0750 0.1472 0.2639 −0.4250
Standard error 0.1782 0.1782 0.1782 0.1782 0.1782 0.1782 0.1782
Alcohol productionb
T value
P value
Effect
Standard error
T value
P value
9.14 −4.95 −2.66 −0.21 0.41 0.74 −1.19
0.001c 0.008c
−0.2667 −0.5222 −0.1667 0.1222 0.0111 0.1111 −0.1111
0.04615 0.04615 0.04615 0.04615 0.04615 0.04615 0.04615
−2.89 −5.66 −1.81 1.32 0.12 1.20 −1.20
0.045c 0.005c 0.145 0.256 0.910 0.295 0.295
0.057 0.844 0.701 0.500 0.299
= 0.9670. = 0.9235. Model terms were significant. X1 : Cereal substrates ratio of malt, rice and maize. X2 : Inoculum amount (%). X3 : Cell ratio of S. cerevisiae and L. helveticus KLDS1.9204. X4 : Fermentation time (h) X5 : Fermentation temperature (°C). X6 , X7 : Dummy factors. a 2 R b 2 R c
a SPX-150B incubator (Zhicheng instruments, Shanghai, China) for 24 h with no pH control. The fermentation substrates were centrifuged at 6000 rpm for 5 min (GL-21M; Centrifugal Machinery Research Institute, Shanghai, China). The pH, titratable acidity, SSC, free amino acids, alcohol concentration, and organic acids of the supernatant were determined.
Analytical methods pH. The pH of the multi-cereal fermented beverage was monitored using a digital pH meter (Delta320; Mettler Toledo, Zurich, Switzerland). Titratable acidity. Titratable acidity was analyzed according to Moneruzzaman and others (2012). Titrable acidity was determined by titrating 10 mL of each sample with 0.1 mol/L NaOH, using phenolphthalein as an indicator. Soluble solids content. SSC (°Brix) was determined at 25 °C with a digital refractometer (Huayunante Technology Co., Ltd, Beijing, China). The operator detects critical angle by noting where a dark-bright boundary falls on an engraved scale and the scale can be calibrated in °Brix. The equipment was calibrated with deionized water before samples were measured. Fermented cereal beverage was prepared under the optimum fermentation conditions determined by SA design and was poured onto a prism of the refractometer and SSC was immediately measured. The measurement was performed three times for each sample.
Free amino acids. Samples for free amino acids analysis were prepared according to the method described by Aro and others (2010). Free amino acid in the supernatant was analyzed using a fully automated amino acid analyzer HITACHI l-8800A (Hitachi Ltd., Tokyo, Japan). The concentration of free amino acids in fermented supernatant was calculated by calibrating with standard amino acids and expressed as mg/L sample. Total reducing sugars. The dinitrosalicylic acid method (DNS) was used to determine total reducing sugars (Moneruzzaman and others 2012). The absorbance of the samples was measured at 540 nm in a DU 800 spectrophotometer (Beckman Coulter Inc., Brea, CA(California), U.S.A.). Alcohol concentration. Alcohol was distilled from the supernatant using a laboratory distillation unit consisting of a distillation flask, a cooler, a flask for collecting alcohol, and a thermometer. Alcohol concentration in the substrates was determined using a hydrometer and expressed in terms of % v/v of alcohol (Dziugan and others 2013). Organic acids. Organic acids of the beverage were determined by HPLC, using an Alliance HPLC system equipped with an Aminex HPX-87H column (300 × 7.8 mm2 ; Bio-Rad, Hercules, California, U.S.A.), and a refractive index detector (Waters 2414) operating at 65 °C, and the reaction mixture was eluted at 0.5 mL/min with 5 mmol/L H2 SO4 . For the quantification of organic acids, calibration curves for each compound were Vol. 80, Nr. 6, 2015 r Journal of Food Science M1261
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Titratable aciditya
Multi-cereal fermented beverage . . . Table 4–Physical–chemical characteristics of mixed cereal sub- °Brix) greatly while total reducing sugars changed little. Sixteen strates and fermented counterpart by L. helveticus KLDS1.9204a . amino acids were detected in the fermented substrates. After ferPhysical– chemical indices
Before fermentation
pH Titratable acidity (°T) Soluble solids content (°Brix) Total reducing sugars (%) Free amino acids (mg/L) Aspartic acid Threonine Serine Glutamic acid Glucine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Arginine Proline lysine Total
5.13 ± 6.61 ± 12.33 ± 8.80 ± 7.5 8.3 5.9 12 2.9 10 –b 11 2.8 6.3 14 8.7 14 3.6 10 30 8.1 160
0.01 0.12 0.58 0.07
mentation, the gross content of free amino acids decreased. This may due to utilization by the microorganism. However, the con3.40 ± 0.01 tent of glycine increased and that of valine, arginine kept stable 26.48 ± 0.56 (Table 4).
After fermentation
6.83 ± 0.29 8.48 ± 0.12 3.5 5.1 4.0 10 3.6 6.7 – 11 2.5 6 13 6.8 13 3.2 10 29 7.1 130
a
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The fermented counterpart was made as follows: malt, rice and maize substrates were mixed in the ratio of 5:3:2, then the substrates was inoculated with 1% L. helveticus KLDS1.9204 and fermented at 37 °C for 12 h. b – means not detected.
performed using pure standards (Sigma, St. Louis, MO, U.S.A.) at different concentrations. All samples were analyzed in triplicate. Waters Empower 3 was used to collect and analyze the data.
Statistical Analysis Experimental designs were carried out using Minitab statistical software 16 (Minitab Inc., State College, Pa., U.S.A.). All analyses were performed in triplicate. Statistical analysis was carried out using SPSS software 20.0 (IBM Corp., New York, N.Y., U.S.A.).
Results Screening of lactobacilli strains The cereal substrates fermented by the different lactobacilli had a pH value between 3.30 and 3.84 and a titratable acidity between 7.96 and 26.48 °T. The average pH and titratable acidity of those substrates was 3.54 and 14.40 °T. L. helveticus KLDS 1.9204 showed the highest acid production among the 27 strains (data not shown). Fermentation characteristics of L. helveticus KLDS1.9204 A lag phase of approximately 2 h was observed after fresh L. helveticus KLDS1.9204 cells were inoculated into multi-cereal substrates. The exponential growth phase lasted for about 8 h before the cells entered stationary phase. Cell concentration at the end of fermentation reached 8.43 log10 CFU/mL. L. helveticus KLDS1.9204 was not able to utilize starch as a sole carbon source. However, it exhibited proteolytic activity on skim milk agar, with a hale zone diameter of 8.08 ± 0.44 mm. The protease activity of the cell-free supernatant of L. helveticus KLDS1.9204 was 84.55 ± 0.51 μ/mL. After fermentation for 12 h, nutrients in the mixed cereal substrate were utilized by L. helveticus KLDS1.9204 and a variety of metabolites were produced. The pH of the mixed cereal substrate decreased from 5.13 to 3.40 and titratable acidity increased correspondently from 6.61 to 26.48 °T. SSC decreased (as measured by M1262 Journal of Food Science r Vol. 80, Nr. 6, 2015
Optimization of fermentation parameters using PB design and SA PB design. The ANOVA of PB design for titratable acidity and alcohol production is shown in Table 3. The coefficient of determination (R2 ) of the first-order model was 0.9670 for titratable acidity and 0.9235 for alcohol production, indicating that the data variability could be explained by the models very well. Usually, a model is considered to be significant when its value of ‘‘P value’’ is less than 0.05. In this case, cereal substrates ratio of malt, rice, and maize and inoculum amount were significant model terms. By applying multiple regression analysis on the experimental data, the following first-order polynomial equations were established to explain titratable acidity and alcohol production: Y1 = 28.0 + 1.63X1 − 0.882X2 − 0.474X3 − 0.038X4 + 0.074X5
(1)
Y2 = 0.822 − 0.133X1 − 0.261X2 − 0.0833X3 + 0.0611X4 − 0.0056X5
(2)
where Y1 represented the titratable acidity, Y2 the alcohol production, X1 the cereal substrate ratio of malt, rice, and maize, X2 the inoculum amount, X3 the cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, X4 the fermentation time, and X5 the fermentation temperature. Therefore, the optimum combination of the variables (cereal substrates ratio, inoculum amount, and cell ratio of S. cerevisiae to L. helveticus KLDS1.9204), which had the highest significant influence on titratable acidity and alcohol production was further analyzed by SA test according to titratable acidity and alcohol production. While the value of those factors should be continuous in SA test, malt substrate content was kept as 50% and maize substrate content was chosen as one of the factors for SA test. Other variables with less significant effect were not included in the next experiment, but instead were used in all trials at their low level and high level, for the negatively contributing variables and the positively contributing variables, respectively. Based on the above results, inoculum, cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, and maize substrate content were the three key factors. The combination of high level of maize substrate content (that is, 25%), the low level of inoculum (that is, 4%), and the medium cell ratio of S. cerevisiae to L. helveticus KLDS1.9204 (that is, 2:1) gave good contribution to titratable acidity and alcohol production in cereal fermentation substrate. The path of SA was needed to minimize the range of values. Steepest ascent. The path of SA was moved along the path in which maize substrate content increased, whereas inoculum and cell ratio of S. cerevisiae to L. helveticus KLDS1.9204 decreased (Table 5). The highest titratable acidity of 29.86 °T and alcohol content of 0.67% was observed under the levels: 5% of inoculum, 2.5:1 of cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, and 25% of maize substrate content.
Multi-cereal fermented beverage . . .
Run
Inoculum amount (%)
Cell ratio of S. cerevisiae to L. helveticus KLDS1.9204
Maize substrate content (%)
Titratable acidity (°T)
Alcohol production (%)
8 7 6 5 4 3 2
4:1 3.5:1 3:1 2.5:1 2:1 1.5:1 1:1
10 15 20 25 30 35 40
26.78 ± 1.58 28.38 ± 0.51 27.66 ± 0.55 29.86 ± 0.90 27.37 ± 2.28 27.36 ± 1.00 26.33 ± 0.36
0.11 ± 0.11 0.29 ± 0.11 0.49 ± 0.11 0.67 ± 0.10 0.62 ± 0.27 0.36 ± 0.09 0.24 ± 0.09
1 2 3 4 5 6 7
Table 6–Physical–chemical characteristics of the end products of a cereal fermented beveragea . Physical–chemical indices Free amino acids (mg/L)
pH Titratable acidity (°T) Soluble solids content (°Brix) Alcohol concentration (%) Aspartic acid Threonine Serine Glutamic acid Glucine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Arginine Proline Lysine Total
End fermented product 3.50 29.86 6.50 0.67 –b 0.3 0.21 0.66 0.86 1.48 0.92 0.31 0.40 – 0.07 1.26 2.59 0.19 0.21 – 0.66 10
a
The fermentation substrate consisted of 50% malt (25 g/100 mL), 25% rice (20 g/100 mL) and 25% maize (30 g/100 mL). The inoculum was 5% with a ration of S. cerevisiae and L. helveticus KLDS1.9204 of 2.5:1. The fermentation temperature was 37 °C and the time was 22 h. Lastly, The fermentation liquors were centrifuged at 6000 rpm for 5 min. b – means not detected.
Evaluation of multi-cereal fermented beverage Multi-cereal fermented beverage manufactured according to the optimum fermentation parameters was evaluated. It had a pH value of 3.5, titratable acidity of 29.86 °T, SSC of 6.5 °Brix, and alcohol concentration of 0.67%. The beverage was rich in organic acids and its contents of oxalic acid, lactic acid, acetic acid, citric acid, and tartaric acid were 1.98, 1.46, 0.65, 0.23, and 0.48 mg/mL, respectively. The gross content of free amino acids was 10 mg/L, including 2.59 mg/L phenylalanine, 1.48 mg/L alanine, and 1.26 mg/L tyrosine (Table 6).
Discussion Cereal was shown to be a good resource for production of novel fermented beverages. Pacala and others (2012) used whole wheat, barley malt, oats, and husked millet for the production of fermented mashes by a mixed culture of mesophilic lactic acid bacteria and wheat beer yeast. As a result, a cereal beverage with good physical–chemical and sensory properties was obtained. As
a substrate for fermentation of beverages, malt is an abundantly available raw material in comparison with milk. In the process of brewing and distilling of malt, some compounds such as organic acids, polyphenols, and lipids also could be extracted. With various kinds of enzymes and rich nutrients, malt has been widely used in fermented beverages (Rozada-S´anchez and others 2008; Rathore and others 2012). Rice is a good source of special fermented flavors, as an ancient alcoholic beverage (rice wine) made of rice is a sweet golden beverage with a harmonious and pleasing fragrance and smooth taste (Shen and others 2012). Luo and others (2008) also found that 13 alcohols, 8 acids, 28 esters, 4 aldehydes and ketones, 17 aromatic compounds, 3 lactones, 6 phenols, 3 sulfides, 9 furans, and 6 nitrogen-containing compounds were identified from typical Chinese rice wine. Maize is a raw material that is widely used in beverage production. Maize contains about 72% starch, 10% protein, and 4% lipid. It also provides many of the B vitamins and essential minerals along with fiber (Ranum and others 2014). Therefore, malt, rice, and maize were chosen as raw materials for production of multi-cereal fermented beverage in this study. Many lactobacilli and bifidobacteria has been used to produce fermented cereal beverages including L. reuteri (Kedia and others 2007), L. plantarum (Coda and others 2012), L. acidophilus (Rathore and others 2012), L. bifermentans (Thapa and Tamang 2004), Bifidobacterium adolescentis, B. infantis, B. Breve, and B. longum (Rozada-S´anchez and others 2008). No studies have examined applicability of L. helveticus in multi-cereal fermented beverage. L. helveticus KLDS1.9204 adapted to the mixed cereal substrate well, and a variety of nutrients could be utilized by the strain including different kinds of fermentable sugars, proteins and amino acids, vitamins, and minerals. During fermentation of mixed cereal substrates by L. helveticus KLDS1.9204, a large amount of acid was produced. Denis and others (1986) also showed that L. helveticus was the most effective strain in whey ultrafiltrate fermentation due to high lactic acid production when compared with other lactic acid bacteria. However, the fermentation characteristics of L. helveticus KLDS1.9204 (such as growth curve, protein digesting capability) requires further analysis. L. helveticus KLDS1.9204 may hydrolyze protein during fermentation. L. helveticus has been reported to have an efficient proteolytic system (Griffiths and Tellez 2013). It is well known that the proteolytic activity of lactobacilli, in terms of the degradation of casein fractions, plays an important role in the development of texture and flavor during the fermentation process (Strahinic and others 2013). Therefore, L. helveticus starters are widely used in the manufacture of fermented milk beverages and hard cheeses (Giraffa and others 1998; Beresford and others 2001; Rossettia and others 2008). Since L. helveticus KLDS1.9204 could not utilize starch as a sole carbon source, some processing technologies were used to
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Table 5–Experimental design and results of the steepest ascend path.
Multi-cereal fermented beverage . . .
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break down starches in the raw materials into monosaccharides or disaccharides. During malting, the barley grain is hydrated and releases hormones which stimulate the secretion of hydrolytic enzymes, including amylases, pentosanases, glucanases, and proteases (Rozada-S´anchez and others 2008). The amylase, glucanase, and protease in the malt could be helpful for starch and protein degradation during saccharification in the water bath. On the other hand, rice and maize substrate were gelatinized by boiling to release the starches from the grains to the substrates, then α-amylase and glucoamylase were added to hydrolyze those starches into fermentable sugars. The ability of lactobacilli species to transform a wide range of carbohydrates into lactic acid is well known (Naveena and others 2005). L. helveticus could utilize different kinds of carbohydrates (almost monosaccharides and disaccharides) in the mixed cereal substrate to produce organic acids (most of which was lactic acid) which induced the increase of titratable acidity and the decrease of pH value. The content and type of organic acids can influence the quality of the end product, which included color, flavor, and stability. The organic acids identified in the end fermented beverage were mainly oxalic acid, lactic acid, acetic acid, citric acid, and tartaric acid. At the same time, proteins in the substrate were hydrolyzed by protease into amino acids which changed the composition and concentrations of amino acids in the fermented mixed cereal. The amino acids can not only provide nitrogen for the growth of microorganisms, but also they can give good color for the beverage. The gross content of free amino acids decreased during fermentation, which may be due to utilization by L. helveticus KLDS1.9204 and yeast, or some amino acids were transformed into alcohol and other metabolites. Different kinds of fermented cereal beverages have been developed. Dlusskaya and others (2008) found that the main fermentation microflora of a commercial kvass sample were L. casei, Leuconostoc mesenteroides and S. cerevisiae. Microbial metabolites in kvass were ethanol, lactate, and acetate, which were the same in the multi-cereal fermented beverage developed in this study. Kedia and others (2007) also developed a fermented malt beverage with the starter culture of L. reuteri and yeast. The malt beverage showed almost the same lactic acid (1.40 mg/mL) as the multi-cereal fermented beverage (1.46 mg/mL), whereas its alcohol content was much higher (1.4%) than the beverage developed in this study (0.67%). The interaction between LAB and yeast is very important, and the mechanism involved has not been widely investigated. Such interaction may stimulate or inhibit the growth of one or both of the two strains. Kedia and others (2007) found that the introduction of the yeast into the malt-based substrates would increased the growth of LAB. The pH of mixed culture broth was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture. The interaction of L. helveticus and S. cerevisia in this study needs further investigate.
Conclusions The results of this study indicate that L. helveticus KLDS1.9204 had the highest acid production among 27 lactobacilli strains tested during fermentation in a multi-cereal substrate and the strain could reach exponential phase after fermentation of 2 h. L. helveticus KLDS1.9204 had a great hydrolyzing capability of proteins; however, it did not show the ability to utilize starch. The content of free amino acids decreased during fermentation of multi-cereal mixture M1264 Journal of Food Science r Vol. 80, Nr. 6, 2015
by L. helveticus KLDS1.9204. Optimum parameters of multi-cereal fermented beverage were: malt substrate (25 g/100 mL) content of 50%, rice (20 g/100 mL) and maize (30 g/100 mL) substrate (30 g/100 mL) contents of 25% respectively, inoculum amount of 5%, cell ratio of S. cerevisiae and L. helveticus KLDS1.9204 of 2.5:1, fermentation temperature of 37 °C, and fermentation time of 22 h. The multi-cereal beverage product had a pH value of 3.5, titratable acidity of 29.86 °T, SSC of 6.5 °Brix, alcohol content of 0.67%, oxalic acid and lactic acid of 1.98 and 1.46 mg/mL, respectively. This fermented cereal beverage has the potential to be developed as a novel beverage. The sensory properties of this product need to be further evaluated.
Acknowledgments This work was supported by Research Fund for the Doctoral Program of Higher Education of China (20122325110017), Outstanding Youth Scientists Foundation of Harbin City, and the Program for Changjiang Scholars and Innovative Research Team in University from China (IRT0959).
Author Contributions Ai Jing performed the experimental work. Ai-Li Li collected test data and analyzed data. Ben-Xian Su and Ai Jing drafted the manuscript. Xiang-Chen Meng designed the study and interpreted the results.
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Multi-cereal fermented beverage . . . Strahinic I, Lozo J, Terzic-Vidojevic A, Fira D, Kojic M, Golic N, Begovic J, Topisirovic L. 2013. Technological and probiotic potential of BGRA43 a natural isolate of Lactobacillus helveticus. Front Microbiol 4:1–5. Thapa S, Tamang JP. 2004. Product characterization of kodo ko jaanr: fermented finger millet beverage of the Himalayas. Food Microbiol 21(5):617–22. Todd EW. 1949. Quantitative studies on the total plasmin and trypsin inhibitor of human blood serum. J Exp Med 39:295–308. Zhu LW, Wang CC, Liu RS, Li HM, Wan DJ, Tang YJ. 2012. Actinobacillus succinogenes ATCC 55618 fermentation medium optimization for the production of succinic acid by response surface methodology. J Biomed Biotechnol 2012:1–9.
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Rimaux T, Riviere A, Illeghems K, Weckx S, De Vuyst L, Leroy F. 2012. Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH. Appl Environ Microbiol 78(14):4874–83. Rossettia L, Fornasaria ME, Gattib M, Lazzib C, Nevianib E, Giraffa G. 2008. Grana Padano cheese whey starters: Microbial composition and strain distribution. Int J Food Microbiol 127(1–2):168–71. Rozada-S´anchez R, Sattur AP, Thomas K, Pandiella SS. 2008. Evaluation of Bifidobacterium spp. for the production of a potentially probiotic malt-based beverage. Process Biochem 43(8):848–54. Shen F, Li FZ, Liu DL, Xu HR, Ying YB, Li BB. 2012. Ageing status characterization of Chinese rice wines using chemical descriptors combined with multivariate data analysis. Food Control 25(2):458–63.
Vol. 80, Nr. 6, 2015 r Journal of Food Science M1265
A novel multi-cereal-based fermented beverage with suitable aroma, flavor, and pH fermented by lactic acid bacteria and Saccharomyces cerevisiae was developed. Twenty-seven lactobacilli strains were screened for acid production (pH and titratable acidity) in a mixture of malt, rice, and maize substrates. It was found that Lactobacillus helveticus KLDS1.9204 had the greatest acid production among 27 lactobacilli tested. The fermentation performance of L. helveticus KLDS1.9204 was also assayed and the fermentation parameters were optimized using Plackett–Burman design and steepest ascent method. L. helveticus KLDS1.9204 showed good proteolytic capability, however, the strain could not utilize starch. The optimum substrate consisted of 50% malt (25 g/100 mL), 25% rice (20 g/100 mL), and 25% maize (30 g/100 mL). The inoculum was 5% with a ratio of S. cerevisiae to L. helveticus KLDS1.9204 of 2.5:1. The optimum temperature was 37 °C and the time was 22 h. Lastly, the quality of the multi-cereal-based fermented beverage was evaluated. This beverage was light yellow, transparent, and it tasted well with a pleasant acid and a unique flavor of cereals. The beverage was rich in free amino acids and organic acids. The pH and titratable acidity of the beverage were 3.5 and 29.86 °T, respectively. The soluble solids content of the beverage was 6.5 °Brix, and the alcohol content was 0.67%.
Abstract:
A novel multi-cereal-based fermented beverage with suitable aroma, flavor, and pH fermented by lactic acid bacteria and yeast was developed. A combination of starters, including Saccharomyces cerevisiae and Lactobacillus helveticus KLDS1.9204, was suitable for fermentation of cereal. There is potential for production in large scale.
Practical Application:
Introduction There is a considerable amount of work done on cerealcontaining foods fermented by lactic acid bacteria or yeast or their combination. Some probiotic beverages were produced by fermentation of cereals, but most of which took single cereal substrates as delivery vehicles for potential probiotic lactic acid bacteria. Gupta and others (2010) have optimized the process for development of an oats beverage fermented by Lactobacillus plantarum. Coda and others (2012) manufactured functional emmer beverages fermented by L. plantarum 6E. Kedia and others (2007) have used mixed cultures (yeast and L. reuteri) for fermentation of cereal beverages. Rathore and others (2012) have also used mixed cereals including barley and malt as substrates for the production of beverage, and L. plantarum and L. acidophilus were used as starter cultures. So far, little work has been reported on the development of multi-cereal beverages fermented by mixed cultures. Cereals have complex nutrient composition and when they are mixed in certain proportions, the properties of the product can be considerably modified. The nutrient availability for microorganisms may change when different cereals are mixed, furthermore, the growth and metabolism of microorganisms in the mixture may change.
MS 20141087 Submitted 6/24/2014, Accepted 3/2/2015. Authors Jing Ai, Ai-Li Li, Ben-Xian Su, and Xiang-Chen Meng are with Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural Univ., Harbin 150030, China. Authors Jing Ai, Ai-Li Li, Ben-Xian Su, and Xiang-Chen Meng are with Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural Univ., Harbin 150030, China. Direct inquiries to author Meng (E-mail: [email protected]).
R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12859 Further reproduction without permission is prohibited
An appropriate selection of substrate composition and microbial strains is necessary to efficiently control the composition of the metabolic end-products. The main aim of this work is to develop a novel multi-cereal (malt, rice, and maize) beverage fermented by lactic acid bacteria and S. cerevisiae. Fermentation performance of the starter strains was also analyzed. Fermentation parameters including compositional cereal substrates (ratio of malt, rice, and maize), inoculum level, ratio of S. cerevisiae to L. helveticus KLDS1.9204, fermentation temperature and fermentation time were optimized. The pH, titratable acidity, soluble solids content (SSC), free amino acids, and organic acids of beverage were determined.
Materials and Methods Cereal fermentation substrates Malt, maize powder, and rice were purchased from local markets. The malt was milled using a FW100 high-speed smashing machine (Taisite Instruments, Tianjin, China) fitted with a 0.5 mm aperture sieve. Malt fermentation substrate was prepared by dissolving 25 g of malt powder in 100 mL of distilled water and the mixture was then kept in a water bath at 65 °C for 2.5 h. The mixture was then filtered using 8 layers of gauze and finally autoclaved at 121 °C for 15 min. Rice and maize fermentation substrates were prepared by dissolving 20 g of rice or 30 g of maize powder in 100 mL of distilled water. Rice and maize substrates were boiled for 20 min by a wire coil heater (Yongguangming Instruments, Beijing, China) and starches in the fermentation substrates were gelatinized. Starches were hydrolyzed by the addition of 0.5% Vol. 80, Nr. 6, 2015 r Journal of Food Science M1259
M: Food Microbiology & Safety
Keywords: Cereal, fermented beverage, Lactobacillus helveticus, Saccharomyces cerevisiae
Multi-cereal fermented beverage . . . (w/w) of α-amylase (20000 μ/g, Zhaodong, China) in a water bath at 90 °C for 1 h. The substrates were cooled to 65 °C and 1% (w/w) of glucoamylase (100000 μ/g, Hunan, China) was added. The substrates were then transferred to a bath at 65 °C for 2 h. Finally, they were filtered and autoclaved at 121 °C for 15 min.
Microbiological propagation Twenty-seven lactobacilli strains obtained from Bacterial Collection (Key Laboratory of Dairy Science, Northeast Agricultural University, Harbin, China) including 17 strains of L. plantarum, 6 strains of L. paracasei, 2 strains of L. rhamnosus, 1 strain of L. helveticus, and 1 strain of L. brevis were propagated for 12 h at 37 °C in deMan Rogosa Sharp (MRS) broth (Nguyen and Loiseau 2007) from frozen stock cultures. Then, the starter culture was obtained by overnight incubation at 37 °C in MRS broth. Dried S. cerevisiae (Anqi yeast, Yi Chang, Hu Bei, China) was grown in 2% sucrose solution for 2 h at room temperature and used for inoculation immediately.
Table 1–Levels of the variables tested in Plackett–Burman design. Variable code X1 X2 X3 X4 X5 X6 ,X7
Variables Cereal substrates ratio of malt, rice and maize Inoculum amount (%) Cell ratio of S. cerevisiae and L. helveticus KLDS1.9204 Fermentation time (h) Fermentation temperature (°C) Dummy factors
Low Low level (−)
High High level (+)
6:3:1
5:3:2
4 2:1
9 4:1
16 25
22 37
M: Food Microbiology & Safety
Composition of mixed cereal substrates and samples fermented by L. helveticus Mixed cereal substrates were obtained by mixing malt substrate, rice substrate, and maize substrate with the ratio of 5:2:3 (v/v). A 1% inoculum of L. helveticus KLDS1.9204 was added to the mixed cereal substrates and incubated for 12 h. Sampling was performed Screening of lactobacilli strains at the end of the fermentation. Some physical–chemical indices Cereal substrates were fermented by each of the 27 lactobacilli of the substrates and the fermented samples, including the pH, strains for 12 h at 37 °C with 1% inoculation. Sampling was done at titratable acidity, SSC, free amino acids, and total reducing sugars, 12 h; then, the pH and titratable acidity of the fermented product were analyzed. were determined. Fermentation characteristics of Lactobacillus helveticus KLDS1.9204 Growth curve. L. helveticus KLDS1.9204 was added to multicereal substrates with a 1% inoculation and incubated at 37 °C for 24 h. Sampling was performed every 2 h. Growth was monitored by determination of colony-forming units (CFU) in the fermented mixture as described by Rimaux and others (2012). Cell counts (CFU/mL) were obtained by preparing 10-fold serial dilutions of the samples in saline (0.9%, wt/vol NaCl solution) and plating appropriate dilutions onto MRS agar. After incubation at 37 °C for 24 h, CFU were enumerated. Protein degrading and carbohydrate hydrolyzing capabilities. Extracellular proteolytic activity and utilization of starch were assessed by using plate count agar with 50% skim milk and starch agar respectively (Blaiotta and others 2012). Overnight cultures in MRS (10 μL) for L. helveticus KLDS1.9204 were dropped onto the agar surfaces and incubated at 37 °C for 48 h. The diameters of the halo zone on the agar plate were then measured. The hydrolyzing capability of the tested strains was classified as positive when the diameters of clear zone were more than 1 mm. Each assay was performed in triplicate. Protease activity. Protease activity was measured using casein as substrate by Folin-phenol method (Beganovic and others 2013). The cell-free supernatant of L. helveticus KLDS1.9204 (1 mL) was added to 1 mL casein solution (2%, w/v in 0.1 mol/L phosphate buffer, pH 7.2) and the reaction mixture was incubated at 40 °C for 10 min. Then, 2 mL of trichloroacetic acid (TCA) solution (0.4 M TCA) was added for stopping the reaction. After incubation at 40 °C for 20 min, the reaction mixture was centrifuged and the TCA soluble peptides in the supernatant fraction were measured by the method of Todd (1949) with tyrosine (National Institute for Food and Drug Control, China) as the reference compound. One unit of protease activity was defined as the amount of enzyme required to release 1 μg of tyrosine per min. M1260 Journal of Food Science r Vol. 80, Nr. 6, 2015
Optimization of fermentation parameters using Plackett–Burman design and steepest ascent Determination of significant variables using PB design. The Plackett–Burman (PB) design (Naveena and others 2005) for 7 variables included: cereal substrates ratio of malt, rice and maize, inoculum amount, cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, fermentation time, fermentation temperature, and two dummy factors at two levels (+1 and −1) (Table 1) was used for screening fermentation parameters that significantly influenced titratable acidity and alcohol production. The levels of these factors were determined in one-factor experiment (data not shown). In the current study, a 12-run PB design was applied to evaluate 7 factors (including 2 dummy variables). All experiments were carried out in triplicate and the averages of titratable acidity and alcohol production were taken as response. In the experimental design, each row represents an experiment and each column represents an independent variable (Table 2). The signs + and – represent the two different levels (high and low) of the independent variable under investigation. Steepest ascent. Steepest ascent (SA) design (Zhu and others 2012) was used to move rapidly toward the neighborhood of the optimum response. The zero level of PB design was identified as the base point of SA path. The step along the path was determined by the estimated coefficient and practical experience. Experiments were performed along the SA path until the response showed no further increase. Table 3 shows the SA used in this study. The experiments were performed to determine a suitable direction by increasing or decreasing the concentrations of variables according to the results of PB design. Evaluation of multi-cereal fermented beverage Malt, rice, and maize substrates were mixed with the ratio obtained in the SA. Then, L. helveticus KLDS1.9204 and S. cerevisiae were inoculated in the mixed cereal substrates and incubated under the optimum fermentation conditions according to the results obtained in the SA. Fermentations were carried out at 37 °C in
Multi-cereal fermented beverage . . . Table 2–The Plackett–Burman design for 7 variables coded values with titratable acidity and alcohol production as response. Run
X1
X2
X3
X4
X5
X6
X7
Titratable acidity (°T)
Alcohol production (%)
1 2 3 4 5 6 7 8 9 10 11 12
1 1 −1 1 −1 −1 −1 1 −1 1 −1 1
1 −1 −1 1 1 1 1 1 −1 −1 −1 −1
−1 1 1 1 1 −1 1 −1 −1 −1 −1 1
1 −1 1 −1 −1 −1 1 1 −1 −1 1 1
−1 −1 1 1 1 −1 −1 1 −1 1 1 −1
−1 −1 −1 1 −1 1 1 −1 −1 1 1 1
−1 1 1 −1 −1 1 1 1 −1 1 −1 −1
29.82 ± 1.11 29.57 ± 1.79 26.60 ± 1.67 28.48 ± 2.96 25.43 ± 1.18 25.63 ± 4.00 25.02 ± 3.69 28.27 ± 1.63 27.47 ± 1.89 31.58 ± 1.02 28.02 ± 0.99 30.00 ± 1.68
0.67 ± 0.16 0.83 ± 0.48 1.00 ± 0.13 0.30 ± 0.11 0.63 ± 0.15 0.70 ± 0.28 0.70 ± 0.49 0.37 ± 0.08 1.10 ± 0.33 1.00 ± 0.44 1.60 ± 0.22 0.97 ± 0.08
X1 : Cereal substrates ratio of malt, rice and maize. X2 : Inoculum amount (%). X3 : Cell ratio of S. cerevisiae and L. helveticus KLDS1.9204. X4 : Fermentation time (h) X5 : Fermentation temperature (°C) X6 , X7 : Dummy factors.
Table 3–Effects of the variables and statistical analysis of the PB design.
Variables X1 X2 X3 X4 X5 X6 X7
Effect 3.2583 −1.7639 −0.9472 −0.0750 0.1472 0.2639 −0.4250
Standard error 0.1782 0.1782 0.1782 0.1782 0.1782 0.1782 0.1782
Alcohol productionb
T value
P value
Effect
Standard error
T value
P value
9.14 −4.95 −2.66 −0.21 0.41 0.74 −1.19
0.001c 0.008c
−0.2667 −0.5222 −0.1667 0.1222 0.0111 0.1111 −0.1111
0.04615 0.04615 0.04615 0.04615 0.04615 0.04615 0.04615
−2.89 −5.66 −1.81 1.32 0.12 1.20 −1.20
0.045c 0.005c 0.145 0.256 0.910 0.295 0.295
0.057 0.844 0.701 0.500 0.299
= 0.9670. = 0.9235. Model terms were significant. X1 : Cereal substrates ratio of malt, rice and maize. X2 : Inoculum amount (%). X3 : Cell ratio of S. cerevisiae and L. helveticus KLDS1.9204. X4 : Fermentation time (h) X5 : Fermentation temperature (°C). X6 , X7 : Dummy factors. a 2 R b 2 R c
a SPX-150B incubator (Zhicheng instruments, Shanghai, China) for 24 h with no pH control. The fermentation substrates were centrifuged at 6000 rpm for 5 min (GL-21M; Centrifugal Machinery Research Institute, Shanghai, China). The pH, titratable acidity, SSC, free amino acids, alcohol concentration, and organic acids of the supernatant were determined.
Analytical methods pH. The pH of the multi-cereal fermented beverage was monitored using a digital pH meter (Delta320; Mettler Toledo, Zurich, Switzerland). Titratable acidity. Titratable acidity was analyzed according to Moneruzzaman and others (2012). Titrable acidity was determined by titrating 10 mL of each sample with 0.1 mol/L NaOH, using phenolphthalein as an indicator. Soluble solids content. SSC (°Brix) was determined at 25 °C with a digital refractometer (Huayunante Technology Co., Ltd, Beijing, China). The operator detects critical angle by noting where a dark-bright boundary falls on an engraved scale and the scale can be calibrated in °Brix. The equipment was calibrated with deionized water before samples were measured. Fermented cereal beverage was prepared under the optimum fermentation conditions determined by SA design and was poured onto a prism of the refractometer and SSC was immediately measured. The measurement was performed three times for each sample.
Free amino acids. Samples for free amino acids analysis were prepared according to the method described by Aro and others (2010). Free amino acid in the supernatant was analyzed using a fully automated amino acid analyzer HITACHI l-8800A (Hitachi Ltd., Tokyo, Japan). The concentration of free amino acids in fermented supernatant was calculated by calibrating with standard amino acids and expressed as mg/L sample. Total reducing sugars. The dinitrosalicylic acid method (DNS) was used to determine total reducing sugars (Moneruzzaman and others 2012). The absorbance of the samples was measured at 540 nm in a DU 800 spectrophotometer (Beckman Coulter Inc., Brea, CA(California), U.S.A.). Alcohol concentration. Alcohol was distilled from the supernatant using a laboratory distillation unit consisting of a distillation flask, a cooler, a flask for collecting alcohol, and a thermometer. Alcohol concentration in the substrates was determined using a hydrometer and expressed in terms of % v/v of alcohol (Dziugan and others 2013). Organic acids. Organic acids of the beverage were determined by HPLC, using an Alliance HPLC system equipped with an Aminex HPX-87H column (300 × 7.8 mm2 ; Bio-Rad, Hercules, California, U.S.A.), and a refractive index detector (Waters 2414) operating at 65 °C, and the reaction mixture was eluted at 0.5 mL/min with 5 mmol/L H2 SO4 . For the quantification of organic acids, calibration curves for each compound were Vol. 80, Nr. 6, 2015 r Journal of Food Science M1261
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Titratable aciditya
Multi-cereal fermented beverage . . . Table 4–Physical–chemical characteristics of mixed cereal sub- °Brix) greatly while total reducing sugars changed little. Sixteen strates and fermented counterpart by L. helveticus KLDS1.9204a . amino acids were detected in the fermented substrates. After ferPhysical– chemical indices
Before fermentation
pH Titratable acidity (°T) Soluble solids content (°Brix) Total reducing sugars (%) Free amino acids (mg/L) Aspartic acid Threonine Serine Glutamic acid Glucine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Arginine Proline lysine Total
5.13 ± 6.61 ± 12.33 ± 8.80 ± 7.5 8.3 5.9 12 2.9 10 –b 11 2.8 6.3 14 8.7 14 3.6 10 30 8.1 160
0.01 0.12 0.58 0.07
mentation, the gross content of free amino acids decreased. This may due to utilization by the microorganism. However, the con3.40 ± 0.01 tent of glycine increased and that of valine, arginine kept stable 26.48 ± 0.56 (Table 4).
After fermentation
6.83 ± 0.29 8.48 ± 0.12 3.5 5.1 4.0 10 3.6 6.7 – 11 2.5 6 13 6.8 13 3.2 10 29 7.1 130
a
M: Food Microbiology & Safety
The fermented counterpart was made as follows: malt, rice and maize substrates were mixed in the ratio of 5:3:2, then the substrates was inoculated with 1% L. helveticus KLDS1.9204 and fermented at 37 °C for 12 h. b – means not detected.
performed using pure standards (Sigma, St. Louis, MO, U.S.A.) at different concentrations. All samples were analyzed in triplicate. Waters Empower 3 was used to collect and analyze the data.
Statistical Analysis Experimental designs were carried out using Minitab statistical software 16 (Minitab Inc., State College, Pa., U.S.A.). All analyses were performed in triplicate. Statistical analysis was carried out using SPSS software 20.0 (IBM Corp., New York, N.Y., U.S.A.).
Results Screening of lactobacilli strains The cereal substrates fermented by the different lactobacilli had a pH value between 3.30 and 3.84 and a titratable acidity between 7.96 and 26.48 °T. The average pH and titratable acidity of those substrates was 3.54 and 14.40 °T. L. helveticus KLDS 1.9204 showed the highest acid production among the 27 strains (data not shown). Fermentation characteristics of L. helveticus KLDS1.9204 A lag phase of approximately 2 h was observed after fresh L. helveticus KLDS1.9204 cells were inoculated into multi-cereal substrates. The exponential growth phase lasted for about 8 h before the cells entered stationary phase. Cell concentration at the end of fermentation reached 8.43 log10 CFU/mL. L. helveticus KLDS1.9204 was not able to utilize starch as a sole carbon source. However, it exhibited proteolytic activity on skim milk agar, with a hale zone diameter of 8.08 ± 0.44 mm. The protease activity of the cell-free supernatant of L. helveticus KLDS1.9204 was 84.55 ± 0.51 μ/mL. After fermentation for 12 h, nutrients in the mixed cereal substrate were utilized by L. helveticus KLDS1.9204 and a variety of metabolites were produced. The pH of the mixed cereal substrate decreased from 5.13 to 3.40 and titratable acidity increased correspondently from 6.61 to 26.48 °T. SSC decreased (as measured by M1262 Journal of Food Science r Vol. 80, Nr. 6, 2015
Optimization of fermentation parameters using PB design and SA PB design. The ANOVA of PB design for titratable acidity and alcohol production is shown in Table 3. The coefficient of determination (R2 ) of the first-order model was 0.9670 for titratable acidity and 0.9235 for alcohol production, indicating that the data variability could be explained by the models very well. Usually, a model is considered to be significant when its value of ‘‘P value’’ is less than 0.05. In this case, cereal substrates ratio of malt, rice, and maize and inoculum amount were significant model terms. By applying multiple regression analysis on the experimental data, the following first-order polynomial equations were established to explain titratable acidity and alcohol production: Y1 = 28.0 + 1.63X1 − 0.882X2 − 0.474X3 − 0.038X4 + 0.074X5
(1)
Y2 = 0.822 − 0.133X1 − 0.261X2 − 0.0833X3 + 0.0611X4 − 0.0056X5
(2)
where Y1 represented the titratable acidity, Y2 the alcohol production, X1 the cereal substrate ratio of malt, rice, and maize, X2 the inoculum amount, X3 the cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, X4 the fermentation time, and X5 the fermentation temperature. Therefore, the optimum combination of the variables (cereal substrates ratio, inoculum amount, and cell ratio of S. cerevisiae to L. helveticus KLDS1.9204), which had the highest significant influence on titratable acidity and alcohol production was further analyzed by SA test according to titratable acidity and alcohol production. While the value of those factors should be continuous in SA test, malt substrate content was kept as 50% and maize substrate content was chosen as one of the factors for SA test. Other variables with less significant effect were not included in the next experiment, but instead were used in all trials at their low level and high level, for the negatively contributing variables and the positively contributing variables, respectively. Based on the above results, inoculum, cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, and maize substrate content were the three key factors. The combination of high level of maize substrate content (that is, 25%), the low level of inoculum (that is, 4%), and the medium cell ratio of S. cerevisiae to L. helveticus KLDS1.9204 (that is, 2:1) gave good contribution to titratable acidity and alcohol production in cereal fermentation substrate. The path of SA was needed to minimize the range of values. Steepest ascent. The path of SA was moved along the path in which maize substrate content increased, whereas inoculum and cell ratio of S. cerevisiae to L. helveticus KLDS1.9204 decreased (Table 5). The highest titratable acidity of 29.86 °T and alcohol content of 0.67% was observed under the levels: 5% of inoculum, 2.5:1 of cell ratio of S. cerevisiae to L. helveticus KLDS1.9204, and 25% of maize substrate content.
Multi-cereal fermented beverage . . .
Run
Inoculum amount (%)
Cell ratio of S. cerevisiae to L. helveticus KLDS1.9204
Maize substrate content (%)
Titratable acidity (°T)
Alcohol production (%)
8 7 6 5 4 3 2
4:1 3.5:1 3:1 2.5:1 2:1 1.5:1 1:1
10 15 20 25 30 35 40
26.78 ± 1.58 28.38 ± 0.51 27.66 ± 0.55 29.86 ± 0.90 27.37 ± 2.28 27.36 ± 1.00 26.33 ± 0.36
0.11 ± 0.11 0.29 ± 0.11 0.49 ± 0.11 0.67 ± 0.10 0.62 ± 0.27 0.36 ± 0.09 0.24 ± 0.09
1 2 3 4 5 6 7
Table 6–Physical–chemical characteristics of the end products of a cereal fermented beveragea . Physical–chemical indices Free amino acids (mg/L)
pH Titratable acidity (°T) Soluble solids content (°Brix) Alcohol concentration (%) Aspartic acid Threonine Serine Glutamic acid Glucine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Arginine Proline Lysine Total
End fermented product 3.50 29.86 6.50 0.67 –b 0.3 0.21 0.66 0.86 1.48 0.92 0.31 0.40 – 0.07 1.26 2.59 0.19 0.21 – 0.66 10
a
The fermentation substrate consisted of 50% malt (25 g/100 mL), 25% rice (20 g/100 mL) and 25% maize (30 g/100 mL). The inoculum was 5% with a ration of S. cerevisiae and L. helveticus KLDS1.9204 of 2.5:1. The fermentation temperature was 37 °C and the time was 22 h. Lastly, The fermentation liquors were centrifuged at 6000 rpm for 5 min. b – means not detected.
Evaluation of multi-cereal fermented beverage Multi-cereal fermented beverage manufactured according to the optimum fermentation parameters was evaluated. It had a pH value of 3.5, titratable acidity of 29.86 °T, SSC of 6.5 °Brix, and alcohol concentration of 0.67%. The beverage was rich in organic acids and its contents of oxalic acid, lactic acid, acetic acid, citric acid, and tartaric acid were 1.98, 1.46, 0.65, 0.23, and 0.48 mg/mL, respectively. The gross content of free amino acids was 10 mg/L, including 2.59 mg/L phenylalanine, 1.48 mg/L alanine, and 1.26 mg/L tyrosine (Table 6).
Discussion Cereal was shown to be a good resource for production of novel fermented beverages. Pacala and others (2012) used whole wheat, barley malt, oats, and husked millet for the production of fermented mashes by a mixed culture of mesophilic lactic acid bacteria and wheat beer yeast. As a result, a cereal beverage with good physical–chemical and sensory properties was obtained. As
a substrate for fermentation of beverages, malt is an abundantly available raw material in comparison with milk. In the process of brewing and distilling of malt, some compounds such as organic acids, polyphenols, and lipids also could be extracted. With various kinds of enzymes and rich nutrients, malt has been widely used in fermented beverages (Rozada-S´anchez and others 2008; Rathore and others 2012). Rice is a good source of special fermented flavors, as an ancient alcoholic beverage (rice wine) made of rice is a sweet golden beverage with a harmonious and pleasing fragrance and smooth taste (Shen and others 2012). Luo and others (2008) also found that 13 alcohols, 8 acids, 28 esters, 4 aldehydes and ketones, 17 aromatic compounds, 3 lactones, 6 phenols, 3 sulfides, 9 furans, and 6 nitrogen-containing compounds were identified from typical Chinese rice wine. Maize is a raw material that is widely used in beverage production. Maize contains about 72% starch, 10% protein, and 4% lipid. It also provides many of the B vitamins and essential minerals along with fiber (Ranum and others 2014). Therefore, malt, rice, and maize were chosen as raw materials for production of multi-cereal fermented beverage in this study. Many lactobacilli and bifidobacteria has been used to produce fermented cereal beverages including L. reuteri (Kedia and others 2007), L. plantarum (Coda and others 2012), L. acidophilus (Rathore and others 2012), L. bifermentans (Thapa and Tamang 2004), Bifidobacterium adolescentis, B. infantis, B. Breve, and B. longum (Rozada-S´anchez and others 2008). No studies have examined applicability of L. helveticus in multi-cereal fermented beverage. L. helveticus KLDS1.9204 adapted to the mixed cereal substrate well, and a variety of nutrients could be utilized by the strain including different kinds of fermentable sugars, proteins and amino acids, vitamins, and minerals. During fermentation of mixed cereal substrates by L. helveticus KLDS1.9204, a large amount of acid was produced. Denis and others (1986) also showed that L. helveticus was the most effective strain in whey ultrafiltrate fermentation due to high lactic acid production when compared with other lactic acid bacteria. However, the fermentation characteristics of L. helveticus KLDS1.9204 (such as growth curve, protein digesting capability) requires further analysis. L. helveticus KLDS1.9204 may hydrolyze protein during fermentation. L. helveticus has been reported to have an efficient proteolytic system (Griffiths and Tellez 2013). It is well known that the proteolytic activity of lactobacilli, in terms of the degradation of casein fractions, plays an important role in the development of texture and flavor during the fermentation process (Strahinic and others 2013). Therefore, L. helveticus starters are widely used in the manufacture of fermented milk beverages and hard cheeses (Giraffa and others 1998; Beresford and others 2001; Rossettia and others 2008). Since L. helveticus KLDS1.9204 could not utilize starch as a sole carbon source, some processing technologies were used to
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Table 5–Experimental design and results of the steepest ascend path.
Multi-cereal fermented beverage . . .
M: Food Microbiology & Safety
break down starches in the raw materials into monosaccharides or disaccharides. During malting, the barley grain is hydrated and releases hormones which stimulate the secretion of hydrolytic enzymes, including amylases, pentosanases, glucanases, and proteases (Rozada-S´anchez and others 2008). The amylase, glucanase, and protease in the malt could be helpful for starch and protein degradation during saccharification in the water bath. On the other hand, rice and maize substrate were gelatinized by boiling to release the starches from the grains to the substrates, then α-amylase and glucoamylase were added to hydrolyze those starches into fermentable sugars. The ability of lactobacilli species to transform a wide range of carbohydrates into lactic acid is well known (Naveena and others 2005). L. helveticus could utilize different kinds of carbohydrates (almost monosaccharides and disaccharides) in the mixed cereal substrate to produce organic acids (most of which was lactic acid) which induced the increase of titratable acidity and the decrease of pH value. The content and type of organic acids can influence the quality of the end product, which included color, flavor, and stability. The organic acids identified in the end fermented beverage were mainly oxalic acid, lactic acid, acetic acid, citric acid, and tartaric acid. At the same time, proteins in the substrate were hydrolyzed by protease into amino acids which changed the composition and concentrations of amino acids in the fermented mixed cereal. The amino acids can not only provide nitrogen for the growth of microorganisms, but also they can give good color for the beverage. The gross content of free amino acids decreased during fermentation, which may be due to utilization by L. helveticus KLDS1.9204 and yeast, or some amino acids were transformed into alcohol and other metabolites. Different kinds of fermented cereal beverages have been developed. Dlusskaya and others (2008) found that the main fermentation microflora of a commercial kvass sample were L. casei, Leuconostoc mesenteroides and S. cerevisiae. Microbial metabolites in kvass were ethanol, lactate, and acetate, which were the same in the multi-cereal fermented beverage developed in this study. Kedia and others (2007) also developed a fermented malt beverage with the starter culture of L. reuteri and yeast. The malt beverage showed almost the same lactic acid (1.40 mg/mL) as the multi-cereal fermented beverage (1.46 mg/mL), whereas its alcohol content was much higher (1.4%) than the beverage developed in this study (0.67%). The interaction between LAB and yeast is very important, and the mechanism involved has not been widely investigated. Such interaction may stimulate or inhibit the growth of one or both of the two strains. Kedia and others (2007) found that the introduction of the yeast into the malt-based substrates would increased the growth of LAB. The pH of mixed culture broth was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture. The interaction of L. helveticus and S. cerevisia in this study needs further investigate.
Conclusions The results of this study indicate that L. helveticus KLDS1.9204 had the highest acid production among 27 lactobacilli strains tested during fermentation in a multi-cereal substrate and the strain could reach exponential phase after fermentation of 2 h. L. helveticus KLDS1.9204 had a great hydrolyzing capability of proteins; however, it did not show the ability to utilize starch. The content of free amino acids decreased during fermentation of multi-cereal mixture M1264 Journal of Food Science r Vol. 80, Nr. 6, 2015
by L. helveticus KLDS1.9204. Optimum parameters of multi-cereal fermented beverage were: malt substrate (25 g/100 mL) content of 50%, rice (20 g/100 mL) and maize (30 g/100 mL) substrate (30 g/100 mL) contents of 25% respectively, inoculum amount of 5%, cell ratio of S. cerevisiae and L. helveticus KLDS1.9204 of 2.5:1, fermentation temperature of 37 °C, and fermentation time of 22 h. The multi-cereal beverage product had a pH value of 3.5, titratable acidity of 29.86 °T, SSC of 6.5 °Brix, alcohol content of 0.67%, oxalic acid and lactic acid of 1.98 and 1.46 mg/mL, respectively. This fermented cereal beverage has the potential to be developed as a novel beverage. The sensory properties of this product need to be further evaluated.
Acknowledgments This work was supported by Research Fund for the Doctoral Program of Higher Education of China (20122325110017), Outstanding Youth Scientists Foundation of Harbin City, and the Program for Changjiang Scholars and Innovative Research Team in University from China (IRT0959).
Author Contributions Ai Jing performed the experimental work. Ai-Li Li collected test data and analyzed data. Ben-Xian Su and Ai Jing drafted the manuscript. Xiang-Chen Meng designed the study and interpreted the results.
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