- Email: [email protected]
Evaluation of fermentation conditions to improve the sensory quality of broomcorn millet sour porridge
LWT - Food Science and Technology 104 (2019) 165–172
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Evaluation of fermentation conditions to improve the sensory quality of broomcorn millet sour porridge
T
Qi Wanga,∗, Cong Liua, Yan-ping Jinga, San-hong Fana, Jin Caib a b
School of Life Science, Shanxi University, Taiyuan, 030006, China Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China
ARTICLE INFO
ABSTRACT
Keywords: Broomcorn millet Sour porridge Fermentation Sensory evaluation
This study aimed to evaluate the use of three strains, namely, Lactobacillus brevis L1, Acetobacter aceti A1 and Saccharomyces cerevisiae E4, as inocula for the fermentation of broomcorn millet sour porridge, and the effect of different conditions on sour porridge fermentation. Results showed that these three strains were all suitable for the broomcorn millet sour porridge fermentation, and the combination of L1, A1 and E4 (1∶1∶1, v/v/v) was the best combination of strains starter. The optimal fermentation conditions were liquid-to-solid ratio of 3.33 (v/w), inoculum size of 4.71% (v/ v), fermentation time of 31.94 h and fermentation temperature of 30.47 °C. In the verification experiment, the sensory evaluation score of sour porridge was extremely close to the predicted value, and the sensory evaluation was the following: milk white, sour smell, millet taste and soft, and the titratable acidity was 74.5 °T. The viable cell counts of bacteria and yeast surviving in the sour porridge were 2.3 × 1012 and 1.4 × 1011 CFU/mL, respectively.
1. Introduction Broomcorn millet (Panicum miliaceum L.), also known as proso millet, panic millet, and wild millet, is an annual herbaceous plant in the genera Panicum L. of the family Gramineae. This crop, which originated from China, has been cultivated for over 7000 years (Wang, Wang, & Wen, 2005), and currently planted mainly in the northern part of China, including the northwest and northeast regions, and in India, Central Europe, and the Middle East (Harlan, 1992). Broomcorn millet contains starch contents similar to other grains (Nuss & Tanumihardjo, 2010; Parameswaran & Sadasivam, 1994), and is a good substrate for malting and fermentation (Zarnkow, Back, Gastl, & Arendt, 2010). In many rural areas of China, such as the western part of Inner Mongolia and the northwestern part of Shanxi, the naturally fermented broomcorn millet sour porridge has been used for human consumption. People introduce the broomcorn millet and water into a jar. Fermentation took place spontaneously due to the natural flora of the broomcorn millet. After a few hours of natural fermentation, the broomcorn millet in the jar could be fermented into sour porridge with a pleasant nutty taste. Various species of lactic acid bacteria (LAB) present in the traditional fermented broomcorn millet sour porridge had been isolated and identified by physiological and biochemical tests (Chen, Yang, Wu, Dai, & Li, 2002). Besides LAB, yeasts and acetic acid bacteria were also present in the naturally fermented broomcorn millet sour porridge (Bai et al., 2010).
∗
The question arose whether the broomcorn millet could be fermented using selected LAB, acetic acid bacteria and yeasts strains as starter cultures (i.e. as inocula) and processed into a sour porridge. As there are no reports on the use of broomcorn millet for non-natural sour porridge fermentation production, the purpose of this research was to evaluate the use of three strains, namely, Lactobacillus brevis L1, Acetobacter aceti A1 and Saccharomyces cerevisiae E4, as inocula for the preparation of broomcorn millet sour porridge and to determine the effects of liquid-to-solid ratio, inoculum size, fermentation time and temperature on this sour porridge fermentation. We also aimed to optimize the fermentation conditions and investigate the chemical, microbial and sensory characteristics of the broomcorn millet sour porridge. This work can provide numerous data for the further industrial production of broomcorn millet sour porridge. Response surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models, evaluating the effects of the factors, and searching for the optimal conditions to achieve desirable responses (Myers & Montgomery, 2002). To our knowledge, RSM has not been applied to study the combined effects of liquid-to-solid ratio, inoculation size, fermentation time and temperature on the fermentation of broomcorn millet sour porridge. Investigating the individual and interactive effects of these fermentation factors will help in evaluating the sensory characteristics of the broomcorn millet sour porridge. In this experiment, the predicted
Corresponding author. School of Life Science, Shanxi University, Taiyuan, 030006, PR China. E-mail address: [email protected] (Q. Wang).
https://doi.org/10.1016/j.lwt.2019.01.037 Received 31 May 2018; Received in revised form 18 January 2019; Accepted 23 January 2019 Available online 23 January 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
response variable was the sensory evaluation score, and the evaluated fermentation factors were liquid-to-solid ratio, inoculum size, fermentation time and temperature.
Table 1 Effect of single-strain fermentation on the titratable acidity and sensory evaluation of broomcorn millet sour porridge.
2. Materials and methods 2.1. Microorganisms The following strains were selected: L. brevis L1 and S. cerevisiae E4 from the Food Microbiology Laboratory, Shanxi University, and A. aceti A1 (ATCC33445) from China General Microbiological Culture Collection Center. Strains L1 and A1 were routinely maintained on MRS (Difco) agar slants, and strain E4 was routinely maintained on potato dextrose agar (PDA, Difco) slants.
Strains
Titratable acidity (°T)
Sensory evaluation scores
Lactobacillus brevis L1 Acetobacter aceti A1 Saccharomyces cerevisiae E4
9.07 ± 0.38b 15.51 ± 3.34c 8.49 ± 0.21a
24.48 ± 2.16b 24.97 ± 0.36b 20 ± 3.12a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05). Table 2 Effect of mixed-strains fermentation on the titratable acidity and sensory evaluation of broomcorn millet sour porridge.
2.2. Inoculum preparation
Strains combinations
Strains L1 and A1 were activated at 30 °C for 24 h in MRS broth medium, whereas strain E4 was activated at 30 °C for 24 h in potato dextrose broth medium. All cultures were diluted with sterile water to obtain inocula containing approximately 1.20 × 1011 CFU/mL for L1 and A1, and 4.50 × 1010 CFU/mL for E4.
L1＋A1 (1∶1, v/v) L1＋E4 (1∶1, v/v) A1＋E4 (1∶1, v/v) L1＋A1＋E4 (1∶1∶1, L1＋A1＋E4 (2∶1∶1, L1＋A1＋E4 (1∶2∶1, L1＋A1＋E4 (1∶1∶2,
2.3. Single-strain fermentation and mixed-strains fermentation
Titratable acidity (°T)
v/v/v) v/v/v) v/v/v) v/v/v)
30.9 34.9 34.7 49.6 50.0 51.7 50.1
± ± ± ± ± ± ±
a
0.11 3.40a 2.79a 1.03b 0.26b 0.01b 1.57b
Sensory evaluation scores 25.20 23.12 23.72 26.28 26.32 24.76 24.66
± ± ± ± ± ± ±
2.72cd 2.20a 1.92ab 4.72d 3.36d 4.71bc 4.84bc
L1＝Lactobacillus brevis, A1＝Acetobacter aceti, E4＝Saccharomyces cerevisiae. Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
For single-strain fermentation, broomcorn millets were purchased on a local market in Hequ, Shanxi Province, China. After cleaning, the broomcorn millet (50 g) and water (150 mL) were introduced into an Erlenmeyer flask. Then, the flasks were heated to 85 °C in a water bath and held for 30 min. After cooling, each flask was inoculated separately with 5% (v/v) inoculum size of each strain. The flasks were incubated in the dark at 30 °C for 24 h, and the acidity and sensory analyses were determined at the end of each process. For the mixed-strains fermentation, similarly, the broomcorn millet (50 g) and water (150 mL) were introduced into an Erlenmeyer flask; then, the flasks were heated at 85 °C for 30 min. In contrast, three strains were mixed according to different combinations. After cooling, each flask was inoculated respectively with 5% (v/v) inoculum size of each combination of strains. The flasks were incubated in the dark at 30 °C for 24 h. The acidity and sensory analyses were determined at the end of each process.
Table 3 Effect of liquid-to-solid ratio on the titratable acidity and sensory evaluation of broomcorn millet sour porridge. Liquid-to-solid ratio (v/w)
Titratable acidity (°T)
2 3 4 5 6
79.44 38.95 34.41 27.29 20.55
± ± ± ± ±
1.35d 0.46c 1.22c 0.30b 1.69a
Sensory evaluation scores 20.96 27.60 19.64 19.16 14.88
± ± ± ± ±
0.72b 0.36c 0.36b 2.12b 0.71a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
2.4. Effect of liquid-to-solid ratio, inoculum size, fermentation temperature and time on sour porridge fermentation
Table 4 Effect of inoculum size on the titratable acidity and sensory evaluation of broomcorn millet sour porridge.
To study the effect of liquid-to-solid ratio on the sour porridge fermentation, 100, 150, 200, 250 and 300 mL of water were added to 50 g of broomcorn millet, resulting in liquid-to-solid ratios of 2, 3, 4, 5 and 6 (v/w), respectively, and the fermentation were carried out at 30 °C for 24 h using 5% (v/v) inoculum size per flask. To study the effect of inoculum size, we conducted a series of fermentation by using 3, 5, 7, 9 and 11% (v/v) inoculum size per flask at 30 °C for 24 h. The effect of fermentation time was studied by a series of fermentations conducted for 6, 12, 18, 24, 30, 36, 42 and 48 h, carried out at 30 °C by employing 5% inoculum size per flask. Meanwhile, the effect of fermentation temperature was investigated by a series of fermentation with 5% inoculum size per flask at 20, 25, 30, 35 and 40 °C respectively for 24 h. Fermentations were performed statically in an incubator set at different temperatures of the experimental design. The acidity and sensory analyses were determined at the end of each process.
Inoculum size (%, v/v)
Titratable acidity (°T)
3 5 7 9 11
61 65 74 70 66
± ± ± ± ±
5.61a 2.79ab 2.77b 2.78ab 4.20ab
Sensory evaluation scores 28.92 33.20 30.84 29.84 27.50
± ± ± ± ±
0.28ab 0.80d 0.24c 0.08bc 0.64a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
by using Minitab software (version 15, USA). The sensory evaluation score of sour porridge, which was used as the response for the fermentation experiment, was assumed to be under the influence of the four tested fermentation conditions, namely, liquid-to-solid ratio, inoculum size, fermentation time and temperature. The range and levels of these variables investigated in this current study were presented in Table 7. For forecasting the optimal point, a second-order polynomial
2.5. Optimization of fermentation RSM using Box-Behnken design (BBD) experiment was applied (Liu & Wang, 2007) to optimize the fermentation conditions. The RSM was designed based on the results of single-factor experiments (Tables 3–6) 166
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
function (Eq. (1)) was fitted to correlate the relationship between the sensory evaluation scores and fermentation conditions.
Table 5 Effect of fermentation time on the titratable acidity and sensory evaluation of broomcorn millet sour porridge. Fermentation time (h)
Titratable acidity (°T)
Sensory evaluation scores
6 12 18 24 30 36 42 48
3.22 ± 0.25a 6.08 ± 0.01b 16.8 ± 1.08c 36.77 ± 1.39d 60 ± 1.44e 66 ± 0.01f 105 ± 0.01g 121 ± 1.76h
19.46 23.22 24.16 28.04 32.20 32.21 28.28 27.74
± ± ± ± ± ± ± ±
Y = β0﹢β1X1﹢β2X2﹢β3X3﹢β4X4﹢β11X12﹢β22X22﹢β33X32﹢β44X42﹢ β12X1X2﹢β13X1X3 ﹢β14X1X4 ﹢β23X2X3﹢β24X2X4 ﹢β34X3X4
0.482a 0.40b 1.56b 0.40c 0.52d 1.72d 0.80c 0.28b
Where Y is the predicted response variable; β0 is the model intercept; β1, β2, β3 and β4 are the linear coefficients; β11, β22, β33 and β44 are the quadratic coefficients; β12, β13, β14, β23, β24 and β34 are the cross-product coefficients; X1, X2, X3 and X4 represent the independent variables; and X1X2 is the interaction term of X1 with X2 (as the name implies), similar to the rest of the variables. The accuracy and general ability of the above equation could be evaluated by the coefficient of determination (R2). Design-Expert 8.0.6 Trial software was employed for regression and graphical analyses of the data. The optimum fermentation conditions of liquid-to-solid ratio, inoculum size, fermentation time, and temperature were obtained by solving the regression equation.
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05). Table 6 Effect of fermentation temperature on the titratable acidity and sensory evaluation of broomcorn millet sour porridge. Fermentation temperature (°C)
Titratable acidity (°T)
Sensory evaluation scores
20 25 30 35 40
7.91 ± 0.01a 39.82 ± 1.48c 52 ± 0.55d 39.81 ± 1.19c 25.60 ± 1.27b
27.40 28 ± 29.95 25.72 24.24
2.6. Chemical analysis of the fermented sour porridge The pH of the fermented sour porridge was measured using a pH meter (Starter 2100; Ohaus, Shanghai, China) fitted with a glass electrode. The titratable acidity (TA) was determined according to the Association of Official Analytical Chemists (1997) Method No.947.05 and expressed as °T. One acidity unit (°T) indicates the amount of 1 mL 0.1 mol equiv/L NaOH used for the titration of 10 mL of sour porridge. Initial pH of the sour porridge was approximately 6.5.
± 1.36c 0.08d ± 0.20e ± 0.68b ± 0.04a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
2.7. Viable cell counts determination Representative 1 mL portions of duplicate sour porridge samples were blended with 9 mL of sterilized saline solution (9 g/100 mL) and subjected to serial dilutions. Total LAB were counted on MRS agar after
Table 7 Box-Behnken design matrix and the response of sensory evaluation scores of broomcorn millet sour porridge. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 a
Liquid-to- solid ratio (v/w) X1
a
2(−1) 4(1) 2(−1) 4(1) 3(0) 3(0) 3(0) 3(0) 2(−1) 4(1) 2(−1) 4(1) 3(0) 3(0) 3(0) 3(0) 2(−1) 4(1) 2(−1) 4(1) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0)
Inoculum size (%,v/v) X2
3(−1) 3(−1) 7(1) 7(1) 5(0) 5(0) 5(0) 5(0) 5(0) 5(0) 5(0) 5(0) 3(−1) 7(1) 3(−1) 7(1) 5(0) 5(0) 5(0) 5(0) 3(−1) 7(1) 3(−1) 7(1) 5(0) 5(0) 5(0) 5(0) 5(0)
(1)
Fermentation time (h) X3
30(0) 30(0) 30(0) 30(0) 24(−1) 36(1) 24(−1) 36(1) 30(0) 30(0) 30(0) 30(0) 24(−1) 24(−1) 36(1) 36(1) 24(−1) 24(−1) 36(1) 36(1) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0)
Fermentation temperature (°C) X4
30(0) 30(0) 30(0) 30(0) 25(−1) 25(−1) 35(1) 35(1) 25(−1) 25(−1) 35(1) 35(1) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 25(−1) 25(−1) 35(1) 35(1) 30(0) 30(0) 30(0) 30(0) 30(0)
(−1), (0) and (1) are coded levels.
167
Sensory evaluation scores (Y) Measured
Predicted
30.84 31 29.60 32.08 27.40 26.48 27.16 28.60 27.76 26.44 26.68 28.40 31.12 31.52 32.08 30.60 31.56 28.56 27.68 33.08 28.44 27.68 29.20 28.72 33.16 33.12 33.12 33.40 33.44
31.35 31.09 29.77 31.84 27.65 26.67 27.23 28.61 27.20 26.59 26.44 28.87 30.92 31.45 32.06 30.71 31.68 28.39 27.68 32.79 28.32 27.77 28.94 28.67 33.25 33.25 33.25 33.25 33.25
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
incubation at 30 °C for 24 h, and yeasts were counted on PDA after incubation at 30 °C for 48 h (Meignen et al., 2001; Wachowskaa U., Irzykowskib W., & Jędryczkab M., 2018). The colonies that appeared on the plates were counted and recorded as colony-forming units (CFU) per mL of sour porridge.
strains combinations of L1, A1 and E4 (2∶1∶1, v/v/v) and L1, A1 and E4 (1∶1∶1, v/v/v). Considering that low inoculation proportions could be economically favorable, the mixed-strains starter combination of L1, A1 and E4 (1∶1∶1, v/v/v) was the best and consequently used as inoculum in the subsequent experiments. Meignen et al. (2001) had also discovered that fermentation with mixed-starters (L. brevis and S. cerevisiae) produced many aroma compounds. The higher production of aroma compounds in the mixed-starter process might be related to the proteolytic activity of LAB (Meignen et al., 2001), which was directly correlated with the sensory characteristics. According to Spicher and Nierle (1988), the amino acids released from dough were used by S. cerevisiae to produce high levels of alcohols. The concentrations of amino acids and aroma compounds were correlated (Torner, MartínezAnaya, Antuña, & Benedito de Barber, 1992). These studies verified that the effects of cereal fermentation with mixed-starter inoculation were better than those with single-strain inoculation.
2.8. Sensory evaluation Quantitative descriptive analysis, based on the method described by the National Standard of the People's Republic of China (2013), was used for the describing the organoleptic properties of the sour porridge. The sensory properties of sour porridges were evaluated by a 10member trained panel (4 men, 6 women; age range 25–53 years) from the School of Life Science in Shanxi University. The samples were served at 4 °C in plastic cups and were coded with three-digit numbers. The order of presentation of samples was randomized. The panel was asked to provide individual scores on a 1−10 scale for color (milk white, light white and dark), a 1−10 scale for flavor (sour smell, light sour smell and bad smell), a 1−10 scale for taste (millet taste, slightly millet taste and bitter taste) and a 1−10 scale for texture (soft, some soft and hard). The sensory evaluation score was the sum of the color, flavor, taste and texture score. The sour porridges were evaluated in three replicates in each session, and the mean score of the sour porridges for each quality attribute was calculated.
3.2. Effects of liquid-to-solid ratio, inoculum size, fermentation time and temperature Liquid-to-solid ratio, inoculum size, fermentation time and fermentation temperature are very important parameters from an industrial perspective. The effects of liquid-to-solid ratio on TA and the sensory evaluation of the broomcorn millet sour porridge were studied at various ratios of 2, 3, 4, 5 and 6 (v/w) (Table 3). The TA had a positive linear relationship with the liquid-to-solid ratio: the lower the ratio, the higher the TA. The highest sensory evaluation score of the sour porridge appeared at the liquid-to-solid ratio of 3 (v/w), and this value was remarkably higher than others (P＜0.05). Therefore, the optimum liquidto-solid ratio was 3 (v/w). The sensory characteristics produced by metabolism of microorganisms could be easily influenced by the water content in cereal-fermented foods, as described by other researchers (Van der Meulen et al., 2007; Wick, Stolz, Böcker, & Lebeault, 2003). Table 4 showed that the inoculum size was not linearly correlated with the TA and sensory evaluation scores. The TA of sour porridges increased from the inoculum size of 3–7% (i.e. 1.035 × 1011 to 2.415 × 1011 CFU/flask). However, the sensory evaluation scores of sour porridges inoculated with the inoculum size of 3, 9 and 11% (i.e. 1.035 × 1011, 3.105 × 1011 and 3.795 × 1011 CFU/flask, respectively) were lower than those of sour porridges inoculated with 5 and 7% (i.e. 1.725 × 1011 and 2.415 × 1011 CFU/flask, respectively). Therefore, the optimum inoculum size was 5% because of its highest sensory evaluation score. Cui, Chen, Wang, and Han (2013) discovered that the effect of inoculum size on the quality of fermented foods was also important. The effects of inoculum size on the volatile compounds directly involved with the sensory characteristics were different for various strains (Helland et al., 2004). During maize porridge fermentation, more lactic acid was significantly produced with higher inoculation rate of L. rhamnosus, and larger inoculation rate of L. acidophilus could result in larger amounts of acetaldehyde and diacetyl. However, the amount of ethanol produced by L. reuteri was independent of the inoculation rate. Furthermore, no significant differences of acetoin concentrations produced by L. rhamnosus were found among different inoculation levels, except after 8 h of fermentation (Helland et al., 2004). Therefore, the inoculum size is overall important to the fermentation result. Table 5 presented that the TA of the sour porridge had a positive linear relationship to the fermentation time: the longer the fermentation time, the higher the TA. The sensory evaluation scores of the sour porridge significantly increased with the prolonging of the fermentation time from 6 h to 36 h, but then decreased with the fermentation time from 42 h to 48 h. Moreover, a slight difference for the sensory evaluation scores was observed between 30 h and 36 h. Therefore, the optimum fermentation time was 30 h. In addition, fermentation time could dramatically influence the fermentation result (Cui et al., 2013; Pereira, Maciel, & Rodrigues, 2011).
2.9. Experimentation and analysis All experiments were replicated in three flasks, and the data were presented as the mean and standard error of three independent experiments. Duncan's multiple range test (Du, 1985) was used to determine the significant differences among mean values at the P＜0.05 level. Analysis of variance of the RSM experimental results was conducted using the Design-Expert 8.0.6 Trial software; the sources of variance were liquid-to-solid ratio, inoculum size, fermentation time and temperature. 3. Results and discussion 3.1. Single-strain fermentation and mixed-strains fermentation Table 1 showed that the TA in strain L1 and E4 was significantly lower than in A1 (P＜0.05). Strains L1 and A1 gave higher sensory evaluation score. Helland, Wicklund, and Narvhus (2004) had discovered that LAB strains could produce different kinds and concentrations of organic acids contributing to sensory evaluation. Some strains of A. aceti were indigenously present in the naturally fermented broomcorn millet sour porridge samples (Bai et al., 2010), and Acetobacter was the second important group of bacteria in food fermentations because it produced acetic acid (Blandinob, Al-Aseeria, Pandiellaa, Canterob, & Webba, 2003). Therefore, strains L1 and A1 were both suitable for the fermentation of broomcorn millet sour porridge. Meanwhile, strain E4 achieved a lower sensory evaluation score compared with strains A1 and L1 (Table 1). However, one of the possible functions of yeasts in fermented foods was the production of aroma compounds (Jespersen, 2003). Thus, strain E4 also contributed to the broomcorn millet sour porridge fermentation. Hence, some sensory characteristics of fermented cereals food including the flavor were caused by the activities of LAB, Acetobacter and yeasts (Blandinob, Al-Aseeria, Pandiellaa, Canterob, & Webba, 2003). Therefore, these three strains L1, A1 and E4 were all useful to the fermentation of broomcorn millet sour porridge. The TA and sensory evaluation of the broomcorn millet sour porridge fermented by mixed-strains were shown in Table 2. Among the seven combinations, the mixed-strains of L. brevis L1, A. aceti A1 and S. cerevisiae E4 in proportions of 1∶1∶1 (v/v/v) and 2∶1∶1 (v/v/v) provided the highest sensory evaluation scores of 26.28 and 26.32, respectively. No significant differences in TA were found between the 168
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
The effect of fermentation temperature on TA and sensory evaluation of the broomcorn millet sour porridge were studied at 20, 25, 30, 35 and 40 °C (Table 6). The temperatures significantly affected the TA and sensory evaluation. The TA of sour porridges fermented at 20 and 40 °C were notably lower than those of sour porridges fermented at 25, 30 and 35 °C. The fermentation temperature of 30 °C resulted in the highest sensory evaluation score of 29.95 and therefore was regarded as the optimum fermentation temperature. According to Cui et al. (2013), the effect of fermentation temperature on walnut milk beverage fermentation was important.
temperature, inoculum size and fermentation time, and fermentation time and fermentation temperature) and the quadratic effects (i.e. liquid-to-solid ratio, inoculum size, fermentation time and fermentation temperature) were significant model factors (Table 8). Furthermore, in the linear effect, the liquid-to-solid ratio with P-value of 0.0002 was the most influencing factor, followed by fermentation temperature and inoculum size. Moreover, the interactive effect of liquid-to-solid ratio and fermentation time had a stronger effect than others. Equation (2) showed that the positive coefficient of linear terms (X1 and X4) and the interactive terms (except X2X3) exerted positive effects to increase the sensory evaluation scores, but the quadratic terms (X12, X22, X32 and X42) had markedly negative effects to the sensory evaluation scores. Using Eq. (2), the 3D response surface plot (Figs. 1–6) to graphically represent the regression equations were generally used to demonstrate the relative effect of liquid-to-solid ratio, inoculum size, fermentation temperature and time. Figs. 1–4 and 6 illustrated that the relative effect could remarkably affect the sensory evaluation scores of sour porridge (P < 0.05), but Fig. 5 indicated the relative effect was insignificant (P > 0.05). The order of most significant effect was listed as follows: interactive term (X1X3, Fig. 2), interactive term (X1X4, Fig. 3), interactive term (X3X4, Fig. 6), interactive term (X1X2, Fig. 1), and interactive term (X2X3, Fig. 4). Furthermore, Fig. 2 showed that the sensory evaluation score of sour porridge was sensitive even when liquid-tosolid ratio and fermentation time were subjected to small alteration, and an increase in sensory evaluation score could be significantly achieved with the increases of liquid-to-solid ratio and fermentation time. The interactive effect of liquid-to-solid ratio and fermentation temperature on the sensory evaluation score of sour porridge was illustrated in Fig. 3; the effect of fermentation temperature on the sensory evaluation score was stronger than that of the liquid-to-solid ratio. The interactive effect of fermentation time and temperature on the sensory evaluation score of sour porridge was illustrated in Fig. 6; the fermentation temperature had a considerably larger effect on the sensory evaluation score than fermentation time. Compared with the results in Fig. 2, the interactive effect of liquid-to-solid ratio and inoculum size on the sensory evaluation score of sour porridge (Fig. 1) was low. The interactive effect of inoculum size and fermentation time on the sensory evaluation score of sour porridge was illustrated in Fig. 4; the effect of inoculum size on the sensory evaluation score was weaker than that of fermentation time. The interactive effect of inoculum size and fermentation temperature on the sensory evaluation score of sour porridge was insignificant (Fig. 5). Such findings conformed to the results in Table 8. Through RSM experiments (Table 7), the theoretical optimum fermentation conditions for the broomcorn millet sour porridge were follows: liquid-to-solid ratio of 3.33 (v/w), inoculum size of 4.71% (v/ v) of the mixed-strains starter (L. brevis L1, A. aceti A1 and S. cerevisiae E4, 1∶1∶1, v/v/v), fermentation time of 31.94 h and fermentation temperature of 30.47 °C. Under these conditions, the sensory evaluation score of the sour porridge reached its maximum value of 33.37. In the verification experiment, the liquid-to-solid ratio, inoculum size of the mixed-strains starter (L. brevis L1, A. aceti A1 and S. cerevisiae E4, 1∶1∶1, v/v/v), fermentation temperature and fermentation time were set as 3 (v/w), 5% (v/v), 30 °C and 30 h, respectively. Under these conditions, the sensory evaluation score of sour porridge was 33.06, which was extremely close to the predicted value, and suggested that the model about sensory evaluation score were adoptable. Under these fermentation conditions, the sensory characteristics of the sour porridge were as follows: milk white, sour smell, millet taste and soft; the TA of the sour porridge was 74.5 °T. According to Fenaroli (1995), the acids were normally characterized as being “sour”. However, in uji (an East African Sour Porridge), the esters contributed to the fruity odors were probably more important for the sensory characteristics than the acids (Masha, Ipsen, Petersen, & Jakobsen, 1998). Other authors found that many alcohols and acids were produced during the fermentation of
3.3. Optimization of fermentation conditions The BBD and corresponding data were listed in Table 7. Regression analysis of the data was performed to test the adequacy of the proposed quadratic model, and the following second-order polynomial equation was derived (Eq. (2)). Y = 33.25﹢0.45X1﹣0.21X2﹢0.10X3 0.76X1X4﹣0.47X2X3 ﹢0.07X2X4 4.28X42
﹢0.38X4 ﹢0.58X1X2 ﹢2.10X1X3 ﹢ ﹢0.59X3X4﹣1.69X12﹣0.54X22﹣1.42X32﹣ (2)
Where Y is the predicted response, and X1, X2, X3 and X4 are the coded values of the liquid-to-solid ratio, inoculum size, fermentation time and temperature, respectively. Table 8 showed the ANOVA for the experiment. The R2 was 0.9908, indicating that the observed and predicted responses were well correlated, and 99.08% of the variability in the response could be explained by the model. For a good statistical model, R2 value should be close to 1.0 where a value of > 0.75 indicates the aptness of the model. Furthermore, the model indicated that the predicted R2 value of 0.9495 was in a reasonable agreement with the adjusted R2 value of 0.9815. The coefficient of variation had a low value of 1.07%, revealing that the model was reliable. Relatively high F-value of 107.31 and extremely low P-value of < 0.0001 indicated that the experimental model excellently conformed to the experiment for the response. The lack of fit was insignificant (P = 0.0596). The model terms X1, X2, X4, X1X2, X1X3, X1X4, X2X3, X3X4, X12, X22, X32, and X42 exhibited a confidence level above 95% (P < 0.05). Thus, the linear effects (i.e. liquid-to-solid ratio, inoculum size and fermentation temperature), the interaction effects (i.e. liquid-to-solid ratio and inoculum size, liquid-to-solid ratio and fermentation time, liquid-to-solid ratio and fermentation Table 8 Analysis of variance for the response surface quadratic model. Variablea
Sum of squares
Degree of freedom
Mean of squares
F-value
P-value
Model X1 X2 X3 X4 X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 X12 X22 X32 X42 Residual Lack of fit Pure error Corrected total
155.72 2.47 0.51 0.12 1.73 1.35 17.64 2.31 0.88 0.02 1.39 18.58 1.91 13.12 118.95 1.45 1.35 0.10 157.17
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 10 4 28
11.12 2.47 0.51 0.12 1.73 1.35 17.64 2.31 0.88 0.02 1.39 18.58 1.91 13.12 118.95 0.10 0.14 0.025
107.31 23.79 4.94 1.16 16.72 12.98 170.18 22.29 8.52 0.19 13.43 179.22 18.41 126.60 1147.59
< 0.0001 0.0002 0.0431 0.3001 0.0011 0.0029 < 0.0001 0.0003 0.0112 0.6703 0.0025 < 0.0001 0.0007 < 0.0001 < 0.0001
5.38
0.0596
a Symbols X1, X2, X3 and X4 denote optimal liquid-to-solid ratio, inoculum size, fermentation time and fermentation temperature, respectively.
169
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
Fig. 1. Response surface plot of the combined effects of liquid-to-solid ratio and inoculum size on the sensory evaluation score of sour porridge.
Fig. 2. Response surface plot of the combined effects of liquid-to-solid ratio and fermentation time on the sensory evaluation score of sour porridge.
Fig. 3. Response surface plot of the combined effects of liquid-to-solid ratio and fermentation temperature on the sensory evaluation score of sour porridge.
culture of LAB), the amounts of LAB and yeasts were both above 108 and 106 CFU/g, respectively. In the wheat and spelt laboratory sourdough fermentations inoculated with LAB including L. brevis, the LAB counts reached up to 108–109 CFU/g after the first day of fermentation and slightly changed during the following days; yeasts developed more slowly than LAB and usually reached 107 CFU/g after 4–5 days of backslopping (Van der Meulen et al., 2007). The L. brevis growth in dough
maize, but no esters existed in the fermented products (Halm, Lillie, Sorensen, & Jakobsen, 1993). In the verification experiment, the viable cell counts of bacteria and yeast surviving in the sour porridge were 2.3 × 1012 and 1.4 × 1011 CFU/mL, respectively, which were equal to or higher than the following results. As described by Masha et al. (1998), in uji fermented by three methods (spontaneous, backslopping and a starter 170
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
Fig. 4. Response surface plot of the combined effects of inoculum size and fermentation time on the sensory evaluation score of sour porridge.
Fig. 5. Response surface plot of the combined effects of inoculum size and fermentation temperature on the sensory evaluation score of sour porridge.
Fig. 6. Response surface plot of the combined effects of fermentation time and temperature on the sensory evaluation score of sour porridge.
fermentation inoculated with L. brevis and baker's yeast was faster than that of S. cerevisiae, and L. brevis and S. cerevisiae reached 9.3 × 108 and 5 × 106 CFU/g after fermentation of 20 h (Meignen et al., 2001). Probiotics represent probably the archetypal functional food, and are defined as alive microbial supplements, which beneficially affect the hosts by improving their intestinal microbial balance (Brown & Valiere, 2004; Kalliomaki et al., 2001). According to Espinoza and Navarro (2010), probiotics have been added to yogurt and other fermented dairy products. However, an increased demand for non-dairy probiotic products comes from veganism. Probiotic products from various food matrices, including fruits (Prado, Parada, Pandey, &
Soccol, 2008) and vegetables (Yoon, Woodams, & Hang, 2006), were being developed. In recent decades, several researchers have tried to ferment cereals using various species of LAB to produce cereals-based probiotic foods (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003; Correia, Nunes, Guedes, Barros, & Delgadillo, 2010; Humblot et al., 2014; Mugula, Nnko, Narvhus, & Sorhaug, 2003; Tou et al., 2006). Although some strains of A. aceti and S. cerevisiae were previously detected in the naturally fermented broomcorn millet sour porridge samples (Bai et al., 2010), studies on their application in the fermentation for producing cereals-based probiotic foods have not been reported. The results from this current experiments (Table 2) showed that 171
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
the sensory evaluation score of broomcorn millet sour porridge co-fermented by L. brevis L1, A. aceti A1 and S. cerevisiae E4 was higher than that of single-strain fermentation. The question arises as to how the cofermentation of several different strains can produce such an effect. At this point no satisfactory explanation can be offered. Microorganisms alter the taste of food positively or negatively by producing by-products such as lactic acid, alcohol, acetic acid, CO2 and diacetyl (Klaenhammer & Kullen, 1999). It will be important to assess the performance of these organisms under controlled fermentation and their contribution to the taste and flavor of the product. Further studies may be required to determine the organic acids, volatile aromatic compounds and essential amino acids contents of fermented products to explain the metabolism of selected strains in broomcorn millet sour porridge fermentation.
Halm, M., Lillie, A., Sorensen, A. K., & Jakobsen, M. (1993). Microbiological and aromatic characteristics of fermented maize doughs for kenkey production in Ghana. International Journal of Food Microbiology, 19, 135–143. Harlan, J. R. (1992). Crops and man. Madison, Wis: American Society of Agronomy and Crop Science Society of America. Helland, M. H., Wicklund, T., & Narvhus, J. A. (2004). Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley. International Journal of Food Microbiology, 91, 305–313. Humblot, C., Turpin, W., Chevalier, F., Picq, C., Rochette, I., & Guyot, J. P. (2014). Determination of expression and activity of genes involved in starch metabolism in Lactobacillus plantarum A6 during fermentation of a cereal-based gruel. International Journal of Food Microbiology, 185, 103–111. Jespersen, L. (2003). Occurrence and taxonomical characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages. FEMS Yeast Research, 3, 191–200. Kalliomaki, M., Salminen, S., Arvilommi, H., Kero, P., Koskinen, P., & Isolauri, E. (2001). Probiotics in primary prevention of atopic disease: A randomized placebo controlled trial. Lancet, 357, 1076–1079. Klaenhammer, T. R., & Kullen, M. J. (1999). Selection and design of probiotics. International Journal of Food Microbiology, 50, 45–57. Liu, G. Q., & Wang, X. L. (2007). Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by Agaricus blazei. Applied Microbiology and Biotechnology, 74, 78. Masha, G. G. K., Ipsen, R., Petersen, M. A., & Jakobsen, M. (1998). Microbiological, rheological and aromatic characteristics of fermented Uji (an east African sour porridge). World Journal of Microbiology and Biotechnology, 14, 451–456. Meignen, B., Onno, B., Gélinas, P., Infantes, M., Guilois, S., & Cahagnier, B. (2001). Optimization of sourdough fermentation with Lactobacillus brevis and baker's yeast. Food Microbiology, 18, 239–245. Mugula, J. K., Nnko, S. A. M., Narvhus, J. A., & Sorhaug, T. (2003). Microbiological and fermentation characteristics of togwa, a Tanzanian fermented food. International Journal of Food Microbiology, 80, 187–199. Myers, R. H., & Montgomery, D. C. (2002). Response surface methodology: Process and product optimization using designed experiments. New York: John Wiley and Sons. National Standard of the People’s Republic of China (NSPRC) (2013). GB/T 29605-2013, Sensory analysis-Guide for food sensory quality control. Beijing: Standards Press of China. Nuss, E. T., & Tanumihardjo, S. A. (2010). Maize: A paramount staple crop in the context of global nutrition. Comprehensive Reviews in Food Science and Food Safety, 9, 417–436. Parameswaran, K. P., & Sadasivam, S. (1994). Changes in the carbohydrates and nitrogenous components during germination of proso millet, Panicum miliaecum. Plant Foods for Human Nutrition, 45, 97–102. Pereira, A. L. F., Maciel, T. C., & Rodrigues, S. (2011). Probiotic beverage from cashew apple juice fermented with Lactobacillus casei. Food Research International, 44, 1276–1283. Prado, F. C., Parada, J. L., Pandey, A., & Soccol, C. R. (2008). Trends in non-dairy probiotic beverages. Food Research International, 41, 111–123. Spicher, G., & Nierle, W. (1988). Proteolytic activity of sourdough bacteria. Applied Microbiology and Biotechnology, 28, 487–492. Torner, M. J., Martínez-Anaya, M. A., Antuña, B., & Benedito de Barber, C. (1992). Headspace flavour compounds produced by yeasts and lactobacilli during fermentation of preferments and bread doughs. Internationl Journal of Food Microbiology, 15, 145–152. Tou, E. H., Guyot, J. P., Mouquet-Rivier, C., Rochette, I., Counil, E., Traoré, A. S., et al. (2006). Study through surveys and fermentation kinetics of the traditional processing of pearl millet (Pennisetum glaucum) into ben-saalga, a fermented gruel from Burkina Faso. International Journal of Food Microbiology, 106, 52–60. Van der Meulen, R., Scheirlinck, I., Van Schoor, A., Huys, G., Vancanneyt, M., Vandamme, P., et al. (2007). Population dynamics and metabolite target analysis of lactic acid bacteria during laboratory fermentations of wheat and spelt sourdoughs. Applied and Environmental Microbiology, 73, 4741–4750. Wachowskaa, U., Irzykowskib, W., & Jędryczkab, M. (2018). Agrochemicals: Effect on genetic resistance in yeasts colonizing winter wheat kernels. Ecotoxicology and Environmental Safety, 162, 77–84. Wang, L., Wang, X. Y., & Wen, Q. F. (2005). Research and utilization of proso millet germplasm resource in China. Journal of Plant Genetic Resources, 6, 471–474. Wick, M., Stolz, P., Böcker, G., & Lebeault, J. M. (2003). Influence of several process parameters on sourdough fermentation. Acta Biotechnologica, 23, 51–61. Yoon, K. Y., Woodams, E. E., & Hang, Y. D. (2006). Production of probiotic cabbage juice by lactic acid bacteria. Bioresource Technology, 97, 1427–1430. Zarnkow, M., Back, W., Gastl, M., & Arendt, E. K. (2010). Impact of proso millet (Panicum miliaceum L.) varieties on malting quality. Journal of the American Society of Brewing Chemists, 68, 152–159.
4. Conclusions This study is the first to report on the fermentation of broomcorn millet sour porridge inoculated with selected strains. The following conclusions can be drawn: (1) all of the three strains tested were necessary to the fermentation of broomcorn millet sour porridge; (2) the strains combination of L. brevis L1, A. aceti A1 and S. cerevisiae E4 (1∶1∶1, v/v/v) can be used to ferment broomcorn millet sour porridge; (3) the suggested optimum fermentation conditions were the following: liquid-to-solid ratio of 3 (v/w), inoculum size of 5% (v/v) of the mixedstrains starter consisting of L. brevis L1, A. aceti A1 and S. cerevisiae E4 (1∶1∶1, v/v/v), fermentation time of 30 h and fermentation temperature of 30 °C. Acknowledgments This work was supported by the Natural Science Foundation of Shanxi Province, China (No. 201701D221178 and 201701D221179), Scientific and Technologial Innovation Programs of Higher Education Institutions of Shanxi Province, China (No. 201802006), National Natural Science Foundation of China (No.31601677), and Scientific Instrument Center of Shanxi University, China. References AOAC (1997). Official method of milk analysis: Official methods of analysis (16th ed.). Washington, DC: Association of Official Analytical Chemists. Bai, M., Wang, J., Qing, M. J., Bao, Q. H., Sun, T. S., & Zhang, H. P. (2010). Occurrence and dominance of yeast in naturally fermented congee in Inner Mongolia of China. Microbiology, 37, 1299–1304. Blandino, A., Al-Aseeri, M. E., Pandiella, S. S., Cantero, D., & Webb, C. (2003). Cerealbased fermented foods and beverages. Food Research International, 36, 527–543. Brown, A. C., & Valiere, A. (2004). Probiotics and medical nutrition therapy. Nutrition in Clinical Care, 7, 56–68. Burdock, G. A. (1994). Fenaroli's handbook of flavor ingredients (3rd ed.). Boca Raton: CRC Press1376. Chen, Z. J., Yang, X. Q., Wu, N., Dai, L. J., & Li, C. G. (2002). Isolation and study of biological properties of lactic acid bacteria from acidic-gruel from Hetao area Inner Mongolia. Journal of Inner Mongolia Agricultural University (Natural Science Edition), 23, 62–65. Correia, I., Nunes, A., Guedes, S., Barros, A. S., & Delgadillo, I. (2010). Screening of lactic acid bacteria potentially useful for sorghum fermentation. Journal of Cereal Science, 52, 9–15. Cui, X. H., Chen, S. J., Wang, Y., & Han, J. R. (2013). Fermentation conditions of walnut milk beverage inoculated with kefir grains. LWT-Food Science and Technology, 50, 349–352. Du, R. J. (1985). Biological statistics. Beijing: Higher Education Press. Espinoza, Y. R., & Navarro, Y. G. (2010). Non-dairy probiotic products. Food Microbiology, 27, 1–10.
172
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Evaluation of fermentation conditions to improve the sensory quality of broomcorn millet sour porridge
T
Qi Wanga,∗, Cong Liua, Yan-ping Jinga, San-hong Fana, Jin Caib a b
School of Life Science, Shanxi University, Taiyuan, 030006, China Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China
ARTICLE INFO
ABSTRACT
Keywords: Broomcorn millet Sour porridge Fermentation Sensory evaluation
This study aimed to evaluate the use of three strains, namely, Lactobacillus brevis L1, Acetobacter aceti A1 and Saccharomyces cerevisiae E4, as inocula for the fermentation of broomcorn millet sour porridge, and the effect of different conditions on sour porridge fermentation. Results showed that these three strains were all suitable for the broomcorn millet sour porridge fermentation, and the combination of L1, A1 and E4 (1∶1∶1, v/v/v) was the best combination of strains starter. The optimal fermentation conditions were liquid-to-solid ratio of 3.33 (v/w), inoculum size of 4.71% (v/ v), fermentation time of 31.94 h and fermentation temperature of 30.47 °C. In the verification experiment, the sensory evaluation score of sour porridge was extremely close to the predicted value, and the sensory evaluation was the following: milk white, sour smell, millet taste and soft, and the titratable acidity was 74.5 °T. The viable cell counts of bacteria and yeast surviving in the sour porridge were 2.3 × 1012 and 1.4 × 1011 CFU/mL, respectively.
1. Introduction Broomcorn millet (Panicum miliaceum L.), also known as proso millet, panic millet, and wild millet, is an annual herbaceous plant in the genera Panicum L. of the family Gramineae. This crop, which originated from China, has been cultivated for over 7000 years (Wang, Wang, & Wen, 2005), and currently planted mainly in the northern part of China, including the northwest and northeast regions, and in India, Central Europe, and the Middle East (Harlan, 1992). Broomcorn millet contains starch contents similar to other grains (Nuss & Tanumihardjo, 2010; Parameswaran & Sadasivam, 1994), and is a good substrate for malting and fermentation (Zarnkow, Back, Gastl, & Arendt, 2010). In many rural areas of China, such as the western part of Inner Mongolia and the northwestern part of Shanxi, the naturally fermented broomcorn millet sour porridge has been used for human consumption. People introduce the broomcorn millet and water into a jar. Fermentation took place spontaneously due to the natural flora of the broomcorn millet. After a few hours of natural fermentation, the broomcorn millet in the jar could be fermented into sour porridge with a pleasant nutty taste. Various species of lactic acid bacteria (LAB) present in the traditional fermented broomcorn millet sour porridge had been isolated and identified by physiological and biochemical tests (Chen, Yang, Wu, Dai, & Li, 2002). Besides LAB, yeasts and acetic acid bacteria were also present in the naturally fermented broomcorn millet sour porridge (Bai et al., 2010).
∗
The question arose whether the broomcorn millet could be fermented using selected LAB, acetic acid bacteria and yeasts strains as starter cultures (i.e. as inocula) and processed into a sour porridge. As there are no reports on the use of broomcorn millet for non-natural sour porridge fermentation production, the purpose of this research was to evaluate the use of three strains, namely, Lactobacillus brevis L1, Acetobacter aceti A1 and Saccharomyces cerevisiae E4, as inocula for the preparation of broomcorn millet sour porridge and to determine the effects of liquid-to-solid ratio, inoculum size, fermentation time and temperature on this sour porridge fermentation. We also aimed to optimize the fermentation conditions and investigate the chemical, microbial and sensory characteristics of the broomcorn millet sour porridge. This work can provide numerous data for the further industrial production of broomcorn millet sour porridge. Response surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models, evaluating the effects of the factors, and searching for the optimal conditions to achieve desirable responses (Myers & Montgomery, 2002). To our knowledge, RSM has not been applied to study the combined effects of liquid-to-solid ratio, inoculation size, fermentation time and temperature on the fermentation of broomcorn millet sour porridge. Investigating the individual and interactive effects of these fermentation factors will help in evaluating the sensory characteristics of the broomcorn millet sour porridge. In this experiment, the predicted
Corresponding author. School of Life Science, Shanxi University, Taiyuan, 030006, PR China. E-mail address: [email protected] (Q. Wang).
https://doi.org/10.1016/j.lwt.2019.01.037 Received 31 May 2018; Received in revised form 18 January 2019; Accepted 23 January 2019 Available online 23 January 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
response variable was the sensory evaluation score, and the evaluated fermentation factors were liquid-to-solid ratio, inoculum size, fermentation time and temperature.
Table 1 Effect of single-strain fermentation on the titratable acidity and sensory evaluation of broomcorn millet sour porridge.
2. Materials and methods 2.1. Microorganisms The following strains were selected: L. brevis L1 and S. cerevisiae E4 from the Food Microbiology Laboratory, Shanxi University, and A. aceti A1 (ATCC33445) from China General Microbiological Culture Collection Center. Strains L1 and A1 were routinely maintained on MRS (Difco) agar slants, and strain E4 was routinely maintained on potato dextrose agar (PDA, Difco) slants.
Strains
Titratable acidity (°T)
Sensory evaluation scores
Lactobacillus brevis L1 Acetobacter aceti A1 Saccharomyces cerevisiae E4
9.07 ± 0.38b 15.51 ± 3.34c 8.49 ± 0.21a
24.48 ± 2.16b 24.97 ± 0.36b 20 ± 3.12a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05). Table 2 Effect of mixed-strains fermentation on the titratable acidity and sensory evaluation of broomcorn millet sour porridge.
2.2. Inoculum preparation
Strains combinations
Strains L1 and A1 were activated at 30 °C for 24 h in MRS broth medium, whereas strain E4 was activated at 30 °C for 24 h in potato dextrose broth medium. All cultures were diluted with sterile water to obtain inocula containing approximately 1.20 × 1011 CFU/mL for L1 and A1, and 4.50 × 1010 CFU/mL for E4.
L1＋A1 (1∶1, v/v) L1＋E4 (1∶1, v/v) A1＋E4 (1∶1, v/v) L1＋A1＋E4 (1∶1∶1, L1＋A1＋E4 (2∶1∶1, L1＋A1＋E4 (1∶2∶1, L1＋A1＋E4 (1∶1∶2,
2.3. Single-strain fermentation and mixed-strains fermentation
Titratable acidity (°T)
v/v/v) v/v/v) v/v/v) v/v/v)
30.9 34.9 34.7 49.6 50.0 51.7 50.1
± ± ± ± ± ± ±
a
0.11 3.40a 2.79a 1.03b 0.26b 0.01b 1.57b
Sensory evaluation scores 25.20 23.12 23.72 26.28 26.32 24.76 24.66
± ± ± ± ± ± ±
2.72cd 2.20a 1.92ab 4.72d 3.36d 4.71bc 4.84bc
L1＝Lactobacillus brevis, A1＝Acetobacter aceti, E4＝Saccharomyces cerevisiae. Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
For single-strain fermentation, broomcorn millets were purchased on a local market in Hequ, Shanxi Province, China. After cleaning, the broomcorn millet (50 g) and water (150 mL) were introduced into an Erlenmeyer flask. Then, the flasks were heated to 85 °C in a water bath and held for 30 min. After cooling, each flask was inoculated separately with 5% (v/v) inoculum size of each strain. The flasks were incubated in the dark at 30 °C for 24 h, and the acidity and sensory analyses were determined at the end of each process. For the mixed-strains fermentation, similarly, the broomcorn millet (50 g) and water (150 mL) were introduced into an Erlenmeyer flask; then, the flasks were heated at 85 °C for 30 min. In contrast, three strains were mixed according to different combinations. After cooling, each flask was inoculated respectively with 5% (v/v) inoculum size of each combination of strains. The flasks were incubated in the dark at 30 °C for 24 h. The acidity and sensory analyses were determined at the end of each process.
Table 3 Effect of liquid-to-solid ratio on the titratable acidity and sensory evaluation of broomcorn millet sour porridge. Liquid-to-solid ratio (v/w)
Titratable acidity (°T)
2 3 4 5 6
79.44 38.95 34.41 27.29 20.55
± ± ± ± ±
1.35d 0.46c 1.22c 0.30b 1.69a
Sensory evaluation scores 20.96 27.60 19.64 19.16 14.88
± ± ± ± ±
0.72b 0.36c 0.36b 2.12b 0.71a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
2.4. Effect of liquid-to-solid ratio, inoculum size, fermentation temperature and time on sour porridge fermentation
Table 4 Effect of inoculum size on the titratable acidity and sensory evaluation of broomcorn millet sour porridge.
To study the effect of liquid-to-solid ratio on the sour porridge fermentation, 100, 150, 200, 250 and 300 mL of water were added to 50 g of broomcorn millet, resulting in liquid-to-solid ratios of 2, 3, 4, 5 and 6 (v/w), respectively, and the fermentation were carried out at 30 °C for 24 h using 5% (v/v) inoculum size per flask. To study the effect of inoculum size, we conducted a series of fermentation by using 3, 5, 7, 9 and 11% (v/v) inoculum size per flask at 30 °C for 24 h. The effect of fermentation time was studied by a series of fermentations conducted for 6, 12, 18, 24, 30, 36, 42 and 48 h, carried out at 30 °C by employing 5% inoculum size per flask. Meanwhile, the effect of fermentation temperature was investigated by a series of fermentation with 5% inoculum size per flask at 20, 25, 30, 35 and 40 °C respectively for 24 h. Fermentations were performed statically in an incubator set at different temperatures of the experimental design. The acidity and sensory analyses were determined at the end of each process.
Inoculum size (%, v/v)
Titratable acidity (°T)
3 5 7 9 11
61 65 74 70 66
± ± ± ± ±
5.61a 2.79ab 2.77b 2.78ab 4.20ab
Sensory evaluation scores 28.92 33.20 30.84 29.84 27.50
± ± ± ± ±
0.28ab 0.80d 0.24c 0.08bc 0.64a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
by using Minitab software (version 15, USA). The sensory evaluation score of sour porridge, which was used as the response for the fermentation experiment, was assumed to be under the influence of the four tested fermentation conditions, namely, liquid-to-solid ratio, inoculum size, fermentation time and temperature. The range and levels of these variables investigated in this current study were presented in Table 7. For forecasting the optimal point, a second-order polynomial
2.5. Optimization of fermentation RSM using Box-Behnken design (BBD) experiment was applied (Liu & Wang, 2007) to optimize the fermentation conditions. The RSM was designed based on the results of single-factor experiments (Tables 3–6) 166
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
function (Eq. (1)) was fitted to correlate the relationship between the sensory evaluation scores and fermentation conditions.
Table 5 Effect of fermentation time on the titratable acidity and sensory evaluation of broomcorn millet sour porridge. Fermentation time (h)
Titratable acidity (°T)
Sensory evaluation scores
6 12 18 24 30 36 42 48
3.22 ± 0.25a 6.08 ± 0.01b 16.8 ± 1.08c 36.77 ± 1.39d 60 ± 1.44e 66 ± 0.01f 105 ± 0.01g 121 ± 1.76h
19.46 23.22 24.16 28.04 32.20 32.21 28.28 27.74
± ± ± ± ± ± ± ±
Y = β0﹢β1X1﹢β2X2﹢β3X3﹢β4X4﹢β11X12﹢β22X22﹢β33X32﹢β44X42﹢ β12X1X2﹢β13X1X3 ﹢β14X1X4 ﹢β23X2X3﹢β24X2X4 ﹢β34X3X4
0.482a 0.40b 1.56b 0.40c 0.52d 1.72d 0.80c 0.28b
Where Y is the predicted response variable; β0 is the model intercept; β1, β2, β3 and β4 are the linear coefficients; β11, β22, β33 and β44 are the quadratic coefficients; β12, β13, β14, β23, β24 and β34 are the cross-product coefficients; X1, X2, X3 and X4 represent the independent variables; and X1X2 is the interaction term of X1 with X2 (as the name implies), similar to the rest of the variables. The accuracy and general ability of the above equation could be evaluated by the coefficient of determination (R2). Design-Expert 8.0.6 Trial software was employed for regression and graphical analyses of the data. The optimum fermentation conditions of liquid-to-solid ratio, inoculum size, fermentation time, and temperature were obtained by solving the regression equation.
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05). Table 6 Effect of fermentation temperature on the titratable acidity and sensory evaluation of broomcorn millet sour porridge. Fermentation temperature (°C)
Titratable acidity (°T)
Sensory evaluation scores
20 25 30 35 40
7.91 ± 0.01a 39.82 ± 1.48c 52 ± 0.55d 39.81 ± 1.19c 25.60 ± 1.27b
27.40 28 ± 29.95 25.72 24.24
2.6. Chemical analysis of the fermented sour porridge The pH of the fermented sour porridge was measured using a pH meter (Starter 2100; Ohaus, Shanghai, China) fitted with a glass electrode. The titratable acidity (TA) was determined according to the Association of Official Analytical Chemists (1997) Method No.947.05 and expressed as °T. One acidity unit (°T) indicates the amount of 1 mL 0.1 mol equiv/L NaOH used for the titration of 10 mL of sour porridge. Initial pH of the sour porridge was approximately 6.5.
± 1.36c 0.08d ± 0.20e ± 0.68b ± 0.04a
Data represent means ± standard deviation (n ＝ 3 for titratable acidity; n ＝ 10 for sensory evaluation scores). Values in the same column with different superscripts are significantly different (P＜0.05).
2.7. Viable cell counts determination Representative 1 mL portions of duplicate sour porridge samples were blended with 9 mL of sterilized saline solution (9 g/100 mL) and subjected to serial dilutions. Total LAB were counted on MRS agar after
Table 7 Box-Behnken design matrix and the response of sensory evaluation scores of broomcorn millet sour porridge. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 a
Liquid-to- solid ratio (v/w) X1
a
2(−1) 4(1) 2(−1) 4(1) 3(0) 3(0) 3(0) 3(0) 2(−1) 4(1) 2(−1) 4(1) 3(0) 3(0) 3(0) 3(0) 2(−1) 4(1) 2(−1) 4(1) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0) 3(0)
Inoculum size (%,v/v) X2
3(−1) 3(−1) 7(1) 7(1) 5(0) 5(0) 5(0) 5(0) 5(0) 5(0) 5(0) 5(0) 3(−1) 7(1) 3(−1) 7(1) 5(0) 5(0) 5(0) 5(0) 3(−1) 7(1) 3(−1) 7(1) 5(0) 5(0) 5(0) 5(0) 5(0)
(1)
Fermentation time (h) X3
30(0) 30(0) 30(0) 30(0) 24(−1) 36(1) 24(−1) 36(1) 30(0) 30(0) 30(0) 30(0) 24(−1) 24(−1) 36(1) 36(1) 24(−1) 24(−1) 36(1) 36(1) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0)
Fermentation temperature (°C) X4
30(0) 30(0) 30(0) 30(0) 25(−1) 25(−1) 35(1) 35(1) 25(−1) 25(−1) 35(1) 35(1) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 25(−1) 25(−1) 35(1) 35(1) 30(0) 30(0) 30(0) 30(0) 30(0)
(−1), (0) and (1) are coded levels.
167
Sensory evaluation scores (Y) Measured
Predicted
30.84 31 29.60 32.08 27.40 26.48 27.16 28.60 27.76 26.44 26.68 28.40 31.12 31.52 32.08 30.60 31.56 28.56 27.68 33.08 28.44 27.68 29.20 28.72 33.16 33.12 33.12 33.40 33.44
31.35 31.09 29.77 31.84 27.65 26.67 27.23 28.61 27.20 26.59 26.44 28.87 30.92 31.45 32.06 30.71 31.68 28.39 27.68 32.79 28.32 27.77 28.94 28.67 33.25 33.25 33.25 33.25 33.25
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
incubation at 30 °C for 24 h, and yeasts were counted on PDA after incubation at 30 °C for 48 h (Meignen et al., 2001; Wachowskaa U., Irzykowskib W., & Jędryczkab M., 2018). The colonies that appeared on the plates were counted and recorded as colony-forming units (CFU) per mL of sour porridge.
strains combinations of L1, A1 and E4 (2∶1∶1, v/v/v) and L1, A1 and E4 (1∶1∶1, v/v/v). Considering that low inoculation proportions could be economically favorable, the mixed-strains starter combination of L1, A1 and E4 (1∶1∶1, v/v/v) was the best and consequently used as inoculum in the subsequent experiments. Meignen et al. (2001) had also discovered that fermentation with mixed-starters (L. brevis and S. cerevisiae) produced many aroma compounds. The higher production of aroma compounds in the mixed-starter process might be related to the proteolytic activity of LAB (Meignen et al., 2001), which was directly correlated with the sensory characteristics. According to Spicher and Nierle (1988), the amino acids released from dough were used by S. cerevisiae to produce high levels of alcohols. The concentrations of amino acids and aroma compounds were correlated (Torner, MartínezAnaya, Antuña, & Benedito de Barber, 1992). These studies verified that the effects of cereal fermentation with mixed-starter inoculation were better than those with single-strain inoculation.
2.8. Sensory evaluation Quantitative descriptive analysis, based on the method described by the National Standard of the People's Republic of China (2013), was used for the describing the organoleptic properties of the sour porridge. The sensory properties of sour porridges were evaluated by a 10member trained panel (4 men, 6 women; age range 25–53 years) from the School of Life Science in Shanxi University. The samples were served at 4 °C in plastic cups and were coded with three-digit numbers. The order of presentation of samples was randomized. The panel was asked to provide individual scores on a 1−10 scale for color (milk white, light white and dark), a 1−10 scale for flavor (sour smell, light sour smell and bad smell), a 1−10 scale for taste (millet taste, slightly millet taste and bitter taste) and a 1−10 scale for texture (soft, some soft and hard). The sensory evaluation score was the sum of the color, flavor, taste and texture score. The sour porridges were evaluated in three replicates in each session, and the mean score of the sour porridges for each quality attribute was calculated.
3.2. Effects of liquid-to-solid ratio, inoculum size, fermentation time and temperature Liquid-to-solid ratio, inoculum size, fermentation time and fermentation temperature are very important parameters from an industrial perspective. The effects of liquid-to-solid ratio on TA and the sensory evaluation of the broomcorn millet sour porridge were studied at various ratios of 2, 3, 4, 5 and 6 (v/w) (Table 3). The TA had a positive linear relationship with the liquid-to-solid ratio: the lower the ratio, the higher the TA. The highest sensory evaluation score of the sour porridge appeared at the liquid-to-solid ratio of 3 (v/w), and this value was remarkably higher than others (P＜0.05). Therefore, the optimum liquidto-solid ratio was 3 (v/w). The sensory characteristics produced by metabolism of microorganisms could be easily influenced by the water content in cereal-fermented foods, as described by other researchers (Van der Meulen et al., 2007; Wick, Stolz, Böcker, & Lebeault, 2003). Table 4 showed that the inoculum size was not linearly correlated with the TA and sensory evaluation scores. The TA of sour porridges increased from the inoculum size of 3–7% (i.e. 1.035 × 1011 to 2.415 × 1011 CFU/flask). However, the sensory evaluation scores of sour porridges inoculated with the inoculum size of 3, 9 and 11% (i.e. 1.035 × 1011, 3.105 × 1011 and 3.795 × 1011 CFU/flask, respectively) were lower than those of sour porridges inoculated with 5 and 7% (i.e. 1.725 × 1011 and 2.415 × 1011 CFU/flask, respectively). Therefore, the optimum inoculum size was 5% because of its highest sensory evaluation score. Cui, Chen, Wang, and Han (2013) discovered that the effect of inoculum size on the quality of fermented foods was also important. The effects of inoculum size on the volatile compounds directly involved with the sensory characteristics were different for various strains (Helland et al., 2004). During maize porridge fermentation, more lactic acid was significantly produced with higher inoculation rate of L. rhamnosus, and larger inoculation rate of L. acidophilus could result in larger amounts of acetaldehyde and diacetyl. However, the amount of ethanol produced by L. reuteri was independent of the inoculation rate. Furthermore, no significant differences of acetoin concentrations produced by L. rhamnosus were found among different inoculation levels, except after 8 h of fermentation (Helland et al., 2004). Therefore, the inoculum size is overall important to the fermentation result. Table 5 presented that the TA of the sour porridge had a positive linear relationship to the fermentation time: the longer the fermentation time, the higher the TA. The sensory evaluation scores of the sour porridge significantly increased with the prolonging of the fermentation time from 6 h to 36 h, but then decreased with the fermentation time from 42 h to 48 h. Moreover, a slight difference for the sensory evaluation scores was observed between 30 h and 36 h. Therefore, the optimum fermentation time was 30 h. In addition, fermentation time could dramatically influence the fermentation result (Cui et al., 2013; Pereira, Maciel, & Rodrigues, 2011).
2.9. Experimentation and analysis All experiments were replicated in three flasks, and the data were presented as the mean and standard error of three independent experiments. Duncan's multiple range test (Du, 1985) was used to determine the significant differences among mean values at the P＜0.05 level. Analysis of variance of the RSM experimental results was conducted using the Design-Expert 8.0.6 Trial software; the sources of variance were liquid-to-solid ratio, inoculum size, fermentation time and temperature. 3. Results and discussion 3.1. Single-strain fermentation and mixed-strains fermentation Table 1 showed that the TA in strain L1 and E4 was significantly lower than in A1 (P＜0.05). Strains L1 and A1 gave higher sensory evaluation score. Helland, Wicklund, and Narvhus (2004) had discovered that LAB strains could produce different kinds and concentrations of organic acids contributing to sensory evaluation. Some strains of A. aceti were indigenously present in the naturally fermented broomcorn millet sour porridge samples (Bai et al., 2010), and Acetobacter was the second important group of bacteria in food fermentations because it produced acetic acid (Blandinob, Al-Aseeria, Pandiellaa, Canterob, & Webba, 2003). Therefore, strains L1 and A1 were both suitable for the fermentation of broomcorn millet sour porridge. Meanwhile, strain E4 achieved a lower sensory evaluation score compared with strains A1 and L1 (Table 1). However, one of the possible functions of yeasts in fermented foods was the production of aroma compounds (Jespersen, 2003). Thus, strain E4 also contributed to the broomcorn millet sour porridge fermentation. Hence, some sensory characteristics of fermented cereals food including the flavor were caused by the activities of LAB, Acetobacter and yeasts (Blandinob, Al-Aseeria, Pandiellaa, Canterob, & Webba, 2003). Therefore, these three strains L1, A1 and E4 were all useful to the fermentation of broomcorn millet sour porridge. The TA and sensory evaluation of the broomcorn millet sour porridge fermented by mixed-strains were shown in Table 2. Among the seven combinations, the mixed-strains of L. brevis L1, A. aceti A1 and S. cerevisiae E4 in proportions of 1∶1∶1 (v/v/v) and 2∶1∶1 (v/v/v) provided the highest sensory evaluation scores of 26.28 and 26.32, respectively. No significant differences in TA were found between the 168
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
The effect of fermentation temperature on TA and sensory evaluation of the broomcorn millet sour porridge were studied at 20, 25, 30, 35 and 40 °C (Table 6). The temperatures significantly affected the TA and sensory evaluation. The TA of sour porridges fermented at 20 and 40 °C were notably lower than those of sour porridges fermented at 25, 30 and 35 °C. The fermentation temperature of 30 °C resulted in the highest sensory evaluation score of 29.95 and therefore was regarded as the optimum fermentation temperature. According to Cui et al. (2013), the effect of fermentation temperature on walnut milk beverage fermentation was important.
temperature, inoculum size and fermentation time, and fermentation time and fermentation temperature) and the quadratic effects (i.e. liquid-to-solid ratio, inoculum size, fermentation time and fermentation temperature) were significant model factors (Table 8). Furthermore, in the linear effect, the liquid-to-solid ratio with P-value of 0.0002 was the most influencing factor, followed by fermentation temperature and inoculum size. Moreover, the interactive effect of liquid-to-solid ratio and fermentation time had a stronger effect than others. Equation (2) showed that the positive coefficient of linear terms (X1 and X4) and the interactive terms (except X2X3) exerted positive effects to increase the sensory evaluation scores, but the quadratic terms (X12, X22, X32 and X42) had markedly negative effects to the sensory evaluation scores. Using Eq. (2), the 3D response surface plot (Figs. 1–6) to graphically represent the regression equations were generally used to demonstrate the relative effect of liquid-to-solid ratio, inoculum size, fermentation temperature and time. Figs. 1–4 and 6 illustrated that the relative effect could remarkably affect the sensory evaluation scores of sour porridge (P < 0.05), but Fig. 5 indicated the relative effect was insignificant (P > 0.05). The order of most significant effect was listed as follows: interactive term (X1X3, Fig. 2), interactive term (X1X4, Fig. 3), interactive term (X3X4, Fig. 6), interactive term (X1X2, Fig. 1), and interactive term (X2X3, Fig. 4). Furthermore, Fig. 2 showed that the sensory evaluation score of sour porridge was sensitive even when liquid-tosolid ratio and fermentation time were subjected to small alteration, and an increase in sensory evaluation score could be significantly achieved with the increases of liquid-to-solid ratio and fermentation time. The interactive effect of liquid-to-solid ratio and fermentation temperature on the sensory evaluation score of sour porridge was illustrated in Fig. 3; the effect of fermentation temperature on the sensory evaluation score was stronger than that of the liquid-to-solid ratio. The interactive effect of fermentation time and temperature on the sensory evaluation score of sour porridge was illustrated in Fig. 6; the fermentation temperature had a considerably larger effect on the sensory evaluation score than fermentation time. Compared with the results in Fig. 2, the interactive effect of liquid-to-solid ratio and inoculum size on the sensory evaluation score of sour porridge (Fig. 1) was low. The interactive effect of inoculum size and fermentation time on the sensory evaluation score of sour porridge was illustrated in Fig. 4; the effect of inoculum size on the sensory evaluation score was weaker than that of fermentation time. The interactive effect of inoculum size and fermentation temperature on the sensory evaluation score of sour porridge was insignificant (Fig. 5). Such findings conformed to the results in Table 8. Through RSM experiments (Table 7), the theoretical optimum fermentation conditions for the broomcorn millet sour porridge were follows: liquid-to-solid ratio of 3.33 (v/w), inoculum size of 4.71% (v/ v) of the mixed-strains starter (L. brevis L1, A. aceti A1 and S. cerevisiae E4, 1∶1∶1, v/v/v), fermentation time of 31.94 h and fermentation temperature of 30.47 °C. Under these conditions, the sensory evaluation score of the sour porridge reached its maximum value of 33.37. In the verification experiment, the liquid-to-solid ratio, inoculum size of the mixed-strains starter (L. brevis L1, A. aceti A1 and S. cerevisiae E4, 1∶1∶1, v/v/v), fermentation temperature and fermentation time were set as 3 (v/w), 5% (v/v), 30 °C and 30 h, respectively. Under these conditions, the sensory evaluation score of sour porridge was 33.06, which was extremely close to the predicted value, and suggested that the model about sensory evaluation score were adoptable. Under these fermentation conditions, the sensory characteristics of the sour porridge were as follows: milk white, sour smell, millet taste and soft; the TA of the sour porridge was 74.5 °T. According to Fenaroli (1995), the acids were normally characterized as being “sour”. However, in uji (an East African Sour Porridge), the esters contributed to the fruity odors were probably more important for the sensory characteristics than the acids (Masha, Ipsen, Petersen, & Jakobsen, 1998). Other authors found that many alcohols and acids were produced during the fermentation of
3.3. Optimization of fermentation conditions The BBD and corresponding data were listed in Table 7. Regression analysis of the data was performed to test the adequacy of the proposed quadratic model, and the following second-order polynomial equation was derived (Eq. (2)). Y = 33.25﹢0.45X1﹣0.21X2﹢0.10X3 0.76X1X4﹣0.47X2X3 ﹢0.07X2X4 4.28X42
﹢0.38X4 ﹢0.58X1X2 ﹢2.10X1X3 ﹢ ﹢0.59X3X4﹣1.69X12﹣0.54X22﹣1.42X32﹣ (2)
Where Y is the predicted response, and X1, X2, X3 and X4 are the coded values of the liquid-to-solid ratio, inoculum size, fermentation time and temperature, respectively. Table 8 showed the ANOVA for the experiment. The R2 was 0.9908, indicating that the observed and predicted responses were well correlated, and 99.08% of the variability in the response could be explained by the model. For a good statistical model, R2 value should be close to 1.0 where a value of > 0.75 indicates the aptness of the model. Furthermore, the model indicated that the predicted R2 value of 0.9495 was in a reasonable agreement with the adjusted R2 value of 0.9815. The coefficient of variation had a low value of 1.07%, revealing that the model was reliable. Relatively high F-value of 107.31 and extremely low P-value of < 0.0001 indicated that the experimental model excellently conformed to the experiment for the response. The lack of fit was insignificant (P = 0.0596). The model terms X1, X2, X4, X1X2, X1X3, X1X4, X2X3, X3X4, X12, X22, X32, and X42 exhibited a confidence level above 95% (P < 0.05). Thus, the linear effects (i.e. liquid-to-solid ratio, inoculum size and fermentation temperature), the interaction effects (i.e. liquid-to-solid ratio and inoculum size, liquid-to-solid ratio and fermentation time, liquid-to-solid ratio and fermentation Table 8 Analysis of variance for the response surface quadratic model. Variablea
Sum of squares
Degree of freedom
Mean of squares
F-value
P-value
Model X1 X2 X3 X4 X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 X12 X22 X32 X42 Residual Lack of fit Pure error Corrected total
155.72 2.47 0.51 0.12 1.73 1.35 17.64 2.31 0.88 0.02 1.39 18.58 1.91 13.12 118.95 1.45 1.35 0.10 157.17
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 10 4 28
11.12 2.47 0.51 0.12 1.73 1.35 17.64 2.31 0.88 0.02 1.39 18.58 1.91 13.12 118.95 0.10 0.14 0.025
107.31 23.79 4.94 1.16 16.72 12.98 170.18 22.29 8.52 0.19 13.43 179.22 18.41 126.60 1147.59
< 0.0001 0.0002 0.0431 0.3001 0.0011 0.0029 < 0.0001 0.0003 0.0112 0.6703 0.0025 < 0.0001 0.0007 < 0.0001 < 0.0001
5.38
0.0596
a Symbols X1, X2, X3 and X4 denote optimal liquid-to-solid ratio, inoculum size, fermentation time and fermentation temperature, respectively.
169
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
Fig. 1. Response surface plot of the combined effects of liquid-to-solid ratio and inoculum size on the sensory evaluation score of sour porridge.
Fig. 2. Response surface plot of the combined effects of liquid-to-solid ratio and fermentation time on the sensory evaluation score of sour porridge.
Fig. 3. Response surface plot of the combined effects of liquid-to-solid ratio and fermentation temperature on the sensory evaluation score of sour porridge.
culture of LAB), the amounts of LAB and yeasts were both above 108 and 106 CFU/g, respectively. In the wheat and spelt laboratory sourdough fermentations inoculated with LAB including L. brevis, the LAB counts reached up to 108–109 CFU/g after the first day of fermentation and slightly changed during the following days; yeasts developed more slowly than LAB and usually reached 107 CFU/g after 4–5 days of backslopping (Van der Meulen et al., 2007). The L. brevis growth in dough
maize, but no esters existed in the fermented products (Halm, Lillie, Sorensen, & Jakobsen, 1993). In the verification experiment, the viable cell counts of bacteria and yeast surviving in the sour porridge were 2.3 × 1012 and 1.4 × 1011 CFU/mL, respectively, which were equal to or higher than the following results. As described by Masha et al. (1998), in uji fermented by three methods (spontaneous, backslopping and a starter 170
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
Fig. 4. Response surface plot of the combined effects of inoculum size and fermentation time on the sensory evaluation score of sour porridge.
Fig. 5. Response surface plot of the combined effects of inoculum size and fermentation temperature on the sensory evaluation score of sour porridge.
Fig. 6. Response surface plot of the combined effects of fermentation time and temperature on the sensory evaluation score of sour porridge.
fermentation inoculated with L. brevis and baker's yeast was faster than that of S. cerevisiae, and L. brevis and S. cerevisiae reached 9.3 × 108 and 5 × 106 CFU/g after fermentation of 20 h (Meignen et al., 2001). Probiotics represent probably the archetypal functional food, and are defined as alive microbial supplements, which beneficially affect the hosts by improving their intestinal microbial balance (Brown & Valiere, 2004; Kalliomaki et al., 2001). According to Espinoza and Navarro (2010), probiotics have been added to yogurt and other fermented dairy products. However, an increased demand for non-dairy probiotic products comes from veganism. Probiotic products from various food matrices, including fruits (Prado, Parada, Pandey, &
Soccol, 2008) and vegetables (Yoon, Woodams, & Hang, 2006), were being developed. In recent decades, several researchers have tried to ferment cereals using various species of LAB to produce cereals-based probiotic foods (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003; Correia, Nunes, Guedes, Barros, & Delgadillo, 2010; Humblot et al., 2014; Mugula, Nnko, Narvhus, & Sorhaug, 2003; Tou et al., 2006). Although some strains of A. aceti and S. cerevisiae were previously detected in the naturally fermented broomcorn millet sour porridge samples (Bai et al., 2010), studies on their application in the fermentation for producing cereals-based probiotic foods have not been reported. The results from this current experiments (Table 2) showed that 171
LWT - Food Science and Technology 104 (2019) 165–172
Q. Wang et al.
the sensory evaluation score of broomcorn millet sour porridge co-fermented by L. brevis L1, A. aceti A1 and S. cerevisiae E4 was higher than that of single-strain fermentation. The question arises as to how the cofermentation of several different strains can produce such an effect. At this point no satisfactory explanation can be offered. Microorganisms alter the taste of food positively or negatively by producing by-products such as lactic acid, alcohol, acetic acid, CO2 and diacetyl (Klaenhammer & Kullen, 1999). It will be important to assess the performance of these organisms under controlled fermentation and their contribution to the taste and flavor of the product. Further studies may be required to determine the organic acids, volatile aromatic compounds and essential amino acids contents of fermented products to explain the metabolism of selected strains in broomcorn millet sour porridge fermentation.
Halm, M., Lillie, A., Sorensen, A. K., & Jakobsen, M. (1993). Microbiological and aromatic characteristics of fermented maize doughs for kenkey production in Ghana. International Journal of Food Microbiology, 19, 135–143. Harlan, J. R. (1992). Crops and man. Madison, Wis: American Society of Agronomy and Crop Science Society of America. Helland, M. H., Wicklund, T., & Narvhus, J. A. (2004). Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley. International Journal of Food Microbiology, 91, 305–313. Humblot, C., Turpin, W., Chevalier, F., Picq, C., Rochette, I., & Guyot, J. P. (2014). Determination of expression and activity of genes involved in starch metabolism in Lactobacillus plantarum A6 during fermentation of a cereal-based gruel. International Journal of Food Microbiology, 185, 103–111. Jespersen, L. (2003). Occurrence and taxonomical characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages. FEMS Yeast Research, 3, 191–200. Kalliomaki, M., Salminen, S., Arvilommi, H., Kero, P., Koskinen, P., & Isolauri, E. (2001). Probiotics in primary prevention of atopic disease: A randomized placebo controlled trial. Lancet, 357, 1076–1079. Klaenhammer, T. R., & Kullen, M. J. (1999). Selection and design of probiotics. International Journal of Food Microbiology, 50, 45–57. Liu, G. Q., & Wang, X. L. (2007). Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by Agaricus blazei. Applied Microbiology and Biotechnology, 74, 78. Masha, G. G. K., Ipsen, R., Petersen, M. A., & Jakobsen, M. (1998). Microbiological, rheological and aromatic characteristics of fermented Uji (an east African sour porridge). World Journal of Microbiology and Biotechnology, 14, 451–456. Meignen, B., Onno, B., Gélinas, P., Infantes, M., Guilois, S., & Cahagnier, B. (2001). Optimization of sourdough fermentation with Lactobacillus brevis and baker's yeast. Food Microbiology, 18, 239–245. Mugula, J. K., Nnko, S. A. M., Narvhus, J. A., & Sorhaug, T. (2003). Microbiological and fermentation characteristics of togwa, a Tanzanian fermented food. International Journal of Food Microbiology, 80, 187–199. Myers, R. H., & Montgomery, D. C. (2002). Response surface methodology: Process and product optimization using designed experiments. New York: John Wiley and Sons. National Standard of the People’s Republic of China (NSPRC) (2013). GB/T 29605-2013, Sensory analysis-Guide for food sensory quality control. Beijing: Standards Press of China. Nuss, E. T., & Tanumihardjo, S. A. (2010). Maize: A paramount staple crop in the context of global nutrition. Comprehensive Reviews in Food Science and Food Safety, 9, 417–436. Parameswaran, K. P., & Sadasivam, S. (1994). Changes in the carbohydrates and nitrogenous components during germination of proso millet, Panicum miliaecum. Plant Foods for Human Nutrition, 45, 97–102. Pereira, A. L. F., Maciel, T. C., & Rodrigues, S. (2011). Probiotic beverage from cashew apple juice fermented with Lactobacillus casei. Food Research International, 44, 1276–1283. Prado, F. C., Parada, J. L., Pandey, A., & Soccol, C. R. (2008). Trends in non-dairy probiotic beverages. Food Research International, 41, 111–123. Spicher, G., & Nierle, W. (1988). Proteolytic activity of sourdough bacteria. Applied Microbiology and Biotechnology, 28, 487–492. Torner, M. J., Martínez-Anaya, M. A., Antuña, B., & Benedito de Barber, C. (1992). Headspace flavour compounds produced by yeasts and lactobacilli during fermentation of preferments and bread doughs. Internationl Journal of Food Microbiology, 15, 145–152. Tou, E. H., Guyot, J. P., Mouquet-Rivier, C., Rochette, I., Counil, E., Traoré, A. S., et al. (2006). Study through surveys and fermentation kinetics of the traditional processing of pearl millet (Pennisetum glaucum) into ben-saalga, a fermented gruel from Burkina Faso. International Journal of Food Microbiology, 106, 52–60. Van der Meulen, R., Scheirlinck, I., Van Schoor, A., Huys, G., Vancanneyt, M., Vandamme, P., et al. (2007). Population dynamics and metabolite target analysis of lactic acid bacteria during laboratory fermentations of wheat and spelt sourdoughs. Applied and Environmental Microbiology, 73, 4741–4750. Wachowskaa, U., Irzykowskib, W., & Jędryczkab, M. (2018). Agrochemicals: Effect on genetic resistance in yeasts colonizing winter wheat kernels. Ecotoxicology and Environmental Safety, 162, 77–84. Wang, L., Wang, X. Y., & Wen, Q. F. (2005). Research and utilization of proso millet germplasm resource in China. Journal of Plant Genetic Resources, 6, 471–474. Wick, M., Stolz, P., Böcker, G., & Lebeault, J. M. (2003). Influence of several process parameters on sourdough fermentation. Acta Biotechnologica, 23, 51–61. Yoon, K. Y., Woodams, E. E., & Hang, Y. D. (2006). Production of probiotic cabbage juice by lactic acid bacteria. Bioresource Technology, 97, 1427–1430. Zarnkow, M., Back, W., Gastl, M., & Arendt, E. K. (2010). Impact of proso millet (Panicum miliaceum L.) varieties on malting quality. Journal of the American Society of Brewing Chemists, 68, 152–159.
4. Conclusions This study is the first to report on the fermentation of broomcorn millet sour porridge inoculated with selected strains. The following conclusions can be drawn: (1) all of the three strains tested were necessary to the fermentation of broomcorn millet sour porridge; (2) the strains combination of L. brevis L1, A. aceti A1 and S. cerevisiae E4 (1∶1∶1, v/v/v) can be used to ferment broomcorn millet sour porridge; (3) the suggested optimum fermentation conditions were the following: liquid-to-solid ratio of 3 (v/w), inoculum size of 5% (v/v) of the mixedstrains starter consisting of L. brevis L1, A. aceti A1 and S. cerevisiae E4 (1∶1∶1, v/v/v), fermentation time of 30 h and fermentation temperature of 30 °C. Acknowledgments This work was supported by the Natural Science Foundation of Shanxi Province, China (No. 201701D221178 and 201701D221179), Scientific and Technologial Innovation Programs of Higher Education Institutions of Shanxi Province, China (No. 201802006), National Natural Science Foundation of China (No.31601677), and Scientific Instrument Center of Shanxi University, China. References AOAC (1997). Official method of milk analysis: Official methods of analysis (16th ed.). Washington, DC: Association of Official Analytical Chemists. Bai, M., Wang, J., Qing, M. J., Bao, Q. H., Sun, T. S., & Zhang, H. P. (2010). Occurrence and dominance of yeast in naturally fermented congee in Inner Mongolia of China. Microbiology, 37, 1299–1304. Blandino, A., Al-Aseeri, M. E., Pandiella, S. S., Cantero, D., & Webb, C. (2003). Cerealbased fermented foods and beverages. Food Research International, 36, 527–543. Brown, A. C., & Valiere, A. (2004). Probiotics and medical nutrition therapy. Nutrition in Clinical Care, 7, 56–68. Burdock, G. A. (1994). Fenaroli's handbook of flavor ingredients (3rd ed.). Boca Raton: CRC Press1376. Chen, Z. J., Yang, X. Q., Wu, N., Dai, L. J., & Li, C. G. (2002). Isolation and study of biological properties of lactic acid bacteria from acidic-gruel from Hetao area Inner Mongolia. Journal of Inner Mongolia Agricultural University (Natural Science Edition), 23, 62–65. Correia, I., Nunes, A., Guedes, S., Barros, A. S., & Delgadillo, I. (2010). Screening of lactic acid bacteria potentially useful for sorghum fermentation. Journal of Cereal Science, 52, 9–15. Cui, X. H., Chen, S. J., Wang, Y., & Han, J. R. (2013). Fermentation conditions of walnut milk beverage inoculated with kefir grains. LWT-Food Science and Technology, 50, 349–352. Du, R. J. (1985). Biological statistics. Beijing: Higher Education Press. Espinoza, Y. R., & Navarro, Y. G. (2010). Non-dairy probiotic products. Food Microbiology, 27, 1–10.
172