- Email: [email protected]
A novel combination of enzymatic hydrolysis and fermentation: Effects on the flavor and nutritional quality of fermented Cordyceps militaris beverage
LWT - Food Science and Technology 120 (2020) 108934
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
A novel combination of enzymatic hydrolysis and fermentation: Effects on the flavor and nutritional quality of fermented Cordyceps militaris beverage
T
Yanyan Laoa,b, Min Zhanga,c,∗, Zhongqin Lid,e, Bhesh Bhandarif a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, Jiangsu, China c Jiangsu Province Key Laboratory of Advanced Food Manufacturing Equipment and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China d Yandi Biological Engineering Co., Ltd, Changde, Hunan, China e Yangzhou Yechun Food Production & Distribution Co, Yangzhou, 225200, Jiangsu, China f School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Enzymatic hydrolysis Two-step fermentation Flavor Nutritional quality Fermented Cordyceps militaris beverage
This study evaluated the effects of enzymatic hydrolysis and fermentation on the flavor and nutritional quality of fermented Cordyceps militaris beverage. Enzymatic hydrolysis can produce more reducing sugars and provide an appropriate fermentation substrate for the growth of bacteria. Electronic nose (e-nose) analysis indicated that first enzymatic hydrolysis and then two-step fermentation could significantly increase the volatile flavor component response values of the fermented beverage. Moreover, electronic tongue (e-tongue) analysis indicated that the fermentation process can increase the sourness and decrease the bitterness of the beverage, resulting in attaining a better taste. Based on the research undertaken on nutritional quality of this fermentation beverage, it was found that a combination of cellulase and pectinase with the ratio of 2:3 yielded higher cordycepin content. After fermentation, the retention rate of cordycepin was also high (98.02%). Fermented beverage pretreated with complex enzymes had a stronger antioxidant capacity compared with non-enzymatic group, which was indicated by the hydroxyl radical scavenging rate and reducing power determination. This fermented C. militaris beverage is expected to help to develop a functional beverage and further research can be carried out on the shelf-life of this beverage.
1. Introduction Cordyceps militaris, food-grade macrofungi belonging to the Ascomycete class, has been widely used as folk tonic food or crude drug in East Asia (Das, Masuda, Sakurai, & Sakakibara, 2010). It is rich in protein, amino acids, and contains diverse biologically active substances such as cordyceps polysaccharides cordycepin, cordycepic acid (D-mannitol) and superoxide dismutase (SOD) (Phan et al., 2018). The main nutrient substance of C. militaris is cordycepin, having various physiological functions such as anti-tumor, hypoglycemic, immunomodulatory, antibacterial and anti-inflammatory (Yang, Gu, & Gu, 2016). It is said that the medicinal value of C. militaris is similar to Cordyceps sinensis and C. militaris are known for their high content of cordycepin (Masuda, Urabe, Sakural, & Sakakibara, 2006). Fresh C. militaris is easy to spoil and can be dried to extend its shelf-life (Zhang et al., 2017). Besides drying, it can also be fermented to delay spoilage (Ma, Zhang, Bhandari, & Gao, 2017; Marsh, Hill, Ross, & Cotter, 2014). Fermented plant extract (FPE) is a new type of fermented beverage ∗
that originated in Japan. FPE is generally made from vegetables, fruits, cereals, legumes, edible fungi (although not plant), which contains abundant nutrients such as enzymes, vitamins, minerals, esters and polyphenols (Feng, Zhang, Mujumdar, & Gao, 2017). FPE is usually fermented by various probiotics, such as lactic acid bacteria, acetic acid bacteria and yeasts (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003). Probiotics are increasingly being used in food products due to their excellent biological activities (Champagne, Gomes da, & Daga, 2018). FPE is different from other fermented products such as wine and yogurt because it contains no alcohol or tiny amount of it (Altay, Karbancioglu-Güler, Daskaya-Dikmen, & Heperkan, 2013), and is not a dairy based food. Edible fungi have high nutrition value of which the content of protein and amino acids, fibers, vitamins, minerals are high, and the fat content is low (Phan et al., 2014; Zhang, Li, & Ding, 2005; Zhang, Zhang, & Shrestha, 2005). There are some research works reported in relation to edible fungi beverage. Hericium erinaceus beverage was found rich in L-glutamic and γ-aminobutyric acid (Woraharn et al., 2016), and shiitake beverage contained important flavor compound
Corresponding author. School of Food Science and Technology, Jiangnan University, 214122 Wuxi, Jiangsu Province, China. E-mail address: [email protected] (M. Zhang).
https://doi.org/10.1016/j.lwt.2019.108934 Received 5 October 2019; Received in revised form 5 December 2019; Accepted 5 December 2019 Available online 07 December 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
preparation process is presented as follows. All treatments were conducted in triplicate. C. militaris pulp preparation: After the C. militaris powder and distilled water mixed in the ratio of 1:10, C. militaris pulp was obtained by intermittent stirring for 2 min (each duration of 10 s) with a high-speed homogenizer (T18BS25; IKA Co., Ltd., Germany) at the rotating speed of 3000–3600 r/min. Enzymatic hydrolysis of C. militaris pulp: Firstly, the pH of C. militaris pulp was adjusted to 4.80 ± 0.05 with 10% w/v food-grade citric acid solution. Then, different ratios of cellulase (0.2%–0.8%, w/v, 50 kU/g, Imperial Jade Biotechnology Co., Ltd., Ningxia, China) and pectinase (0.2%–0.8%, w/v, 100 kU/g, Ruiming Food Additive Co., Ltd., Henan, China) were added. The amount of enzyme should be minimal as it may result in higher production costs (Chong & Wong, 2015). Based on pre-test, the total amount of 1% w/v enzyme was enough. Enzymatic hydrolysis was carried out in a water bath at 50 °C for 4 h. After enzymatic hydrolysis, the enzymes were inactivated at 90 °C for 5 min. Fermentation substrate preparation: 30% w/v C. militaris pulp (with or without enzymatic hydrolysis), 15% w/v brown sugar (Ganzhiyuan sugar industry Co., Ltd., Nanjing, China) and 55% w/v distilled water were mixed uniformly and pasteurized at 85 °C for 10 min. The fermented substrate was cooled to 42 °C and poured into pre-sterilized glass fermenters. Fermentation: The initial pH of the fermentation substrate was adjusted to 4.60–5.00. During the initial phase, the fermentation substrate was inoculated with 0.1% w/v lactic acid bacteria powder (Baishengyou Biotechnology Co., Ltd., Suzhou, China) that contained five strains (Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium lactis and Bifidobacterium longum). The lactic acid fermentation was done at 28 °C for 48 h followed by inoculating 0.1% w/v yeast (Saccharomyces cerevisiae; Angel Yeast Co., Ltd., Hubei, China) to ferment at 28 °C for 12 h. The initial concentration of lactic acid bacteria and yeast S. cerevisiae was 8 × 107 colony-forming units (CFU)/ mL and 5 × 107 CFU/mL, respectively. Fermentation was carried out under anaerobic conditions. After fermentation, the suspension was centrifuged (4200 r/min,
(R)-methyl 2-methyl butanoate up to 35% (Zhang et al., 2018). However, there are only few reports on developing fermented C. militaris beverage. It may be due to the high fiber content of C. millitaris (Phan et al., 2014), which probably affects the fermentation process (Guo et al., 2018). Furthermore, C. militaris has a characteristic odor which is not an acceptable flavor for beverage and needs to be removed or masked by processing. Enzymatic hydrolysis can degrade macromolecules into small molecules (such as low molecular weight sugars) (Sharma, Patel, & Sugandha, 2017). Cellulase and pectinase with the synergistic effect can loosen the structure of materials efficiently due to breakdown of cellulose and pectin into smaller molecules (Lim et al., 2010). This function may hopefully make the water-soluble nutrients (cordycepin) extracted out easily. Besides, the fermentation with probiotics not only can improve the sensory quality but also promote the bioavailability of nutrients (Hancioǧlu & Karapinar, 1997). According to the available literature, the research on the effects of enzymatic hydrolysis and fermentation on C. militaris fermented beverage is rare. Therefore, the purpose of this research work is to study the effects of enzymatic hydrolysis (cellulase and pectinase) and fermentation on pH, titratable acidity, soluble solids (0Brix) and fermenting microorganisms. Moreover, flavor evaluation (e-nose, e-tongue, sensory analysis) and nutritional quality analysis (cordycepin content and antioxidant activity) of C. militaris fermented beverage were carried out, striving to set this fermentation process as a reference for the same category of fermented beverages. 2. Materials and methods 2.1. Materials Dried C. militaris fungi provided by Yandi Biological Engineering Co., Ltd., Hunan, China were smashed through a 100-mesh sieve to obtain the powder and stored in a desiccator before experimentation. 2.2. C. militaris pulp preparation, enzymatic hydrolysis and fermentation The process flow chart is summarized in Fig. 1 and specific
Fig. 1. Flow chart of fermented C. militaris beverage. 2
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
10 min), filtered and sterilized (boiling water bath, 5 min) to obtain the fermented C. militaris beverage. Control: The treatment conditions were the same except that there was no enzymatic hydrolysis treatment of C. militaris pulp.
2.3.7. Measurement of cordycepin content The determination of cordycepin content was according to the protocol of Agricultural Industry Standard of the People's Republic of China (NY/T 2116-2012). Unfermented substrate or fermented beverage (including the experimental group and the control group) was accurately weighed (5 g) and poured into 50 mL volumetric flasks, and then added with about 40 mL of distilled water. After sonication for 30 min, the samples were adjusted to 50 mL with distilled water. Each sample was passed through the 0.45 μm microfiltration membrane. The filtrates were measured by high-performance liquid chromatography (HPLC). Cordycepin content of unfermented substrate (C0) and fermented beverages (C1) were obtained. The retention of cordycepin (%) was calculated as follows:
2.3. Analytical methods 2.3.1. Measurement of the reducing sugars content in C. militaris pulp The content of reducing sugars in C. militaris pulp was determined by 3, 5-dinitrosalicylic acid (DNS) colorimetric method, (Cai, Xiong, Lu, & Sun, 2012). C. militaris pulp was centrifuged (5400 r/min, 20 min) and filtered. The filtrate (1 mL) was mixed with DNS (2 mL) and incubated in the boiling water for 2 min. Then, the mixture was cooled and made up to 25 mL with distilled water. The absorbance was measured at 540 nm. Reducing sugars expressed as grams of equivalent Glu per liter of every sample (g Glu/L). The experiment was performed in triplicate.
Retention of cordycepin (%) = C0/C1 Thermo Fisher Scientific Ultimate3000 system (Thermo Fisher Scientific, Co., Ltd., USA) and a C18 reverse-phase column (250 mm × 4.6 mm i.d., 5 μm) were used to measure the content of cordycepin. The mobile phase consisted of 5% acetonitrile and 95% ultrapure water with a flow rate of 1.0 mL/min. Detection wavelength was set as 260 nm and column temperature was 35 °C. The injection volume of each sample was 10 μL and the detection time was 22 min. The experiment was performed in duplicate.
2.3.2. Measurement of the pH, titratable acidity and soluble solids (0Brix) The pH value was directly measured by a pH meter (Starter 3100; Ohaus instrument Co., Ltd., Shanghai, China) at room temperature. The content of titratable acidity was determined by acid-base titration method following the National Standard of the People's Republic of China (GB/T 12456-2008). A refractometer (WZS-12W) was used to measure soluble solid (0Brix) at 20 °C. The experiment was performed in triplicate.
2.3.8. Hydroxyl radical scavenging rate and reducing power Hydroxyl radical scavenging rate was determined by the salicylic acid method (Smirnoff & Cumbes, 1989). The reaction system contained 1 mL 8.8 mmol/L hydrogen peroxide, 1 mL 9 mmol/L ferrous sulfate, 1 mL 9 mmol/L salicylic acid-ethanol solution, 0.5 mL different treatments fermented beverages with the total volume of the reactants 15 mL (the insufficient volume was made up with distilled water). The final mixture was incubated at 37 °C for 30 min. The absorbance (Ax) was measured with the UV2600 spectrophotometer (Tianmei Scientific Instrument Co., Ltd., Shanghai, China) at 510 nm. Blank control group and another control group were measured as the background absorption of reagent (Ao) and sample (Axo), respectively. The experiment was performed in triplicate.
2.3.3. Enumeration the viable cells of lactic acid bacteria and yeast The viable cells concentration of lactic acid bacteria and yeast S. cerevisiae was determined by plate count method according to the standard of GB 4789.35-2016 and GB 4789.15-2016. All samples were serial tenfold diluted with 0.85% saline solution and were used to inoculate in MRS agar plates for the enumeration of the Lactic acid bacteria and potato dextrose agar plates for the enumeration of the yeast S. cerevisiae. The experiment was performed in duplicate. 2.3.4. E-nose analysis E-nose (iNose; Ruifen Trading Co., Ltd, Shanghai, China) was used to distinguish volatile flavor components of fermented beverages. C. militaris fermentation broth (4 g) was added to the 40 mL sealed vials and left standing at constant temperature (28 °C) for 30 min. The enose, equipped with 14 metal oxide semiconductor gas sensors, has different response values to different smells. Before experiment, the enose needs to be cleaned for 3000 s to ensure the accuracy of subsequent measurement. Measurement stage lasted for 120 s, and gas was delivered to the sensor chamber at the flow rate of 1 L/min. Recovery time (cleaning time) for sensors was 150 s. The experiment was repeated four times.
Hydroxyl radical scavenging rate = [1-(Ax-Axo)/Ao] × 100% A slightly modified method of Oyaizu (1986) was used to analysis the reducing power. Different samples (0.5 mL) were mixed with pH 6.6 phosphate buffer solution (2.5 mL) and 1% w/v potassium ferricyanide solution (2.5 mL). The mixture was heated in a 50 °C water bath for 20 min. After rapidly cooling and centrifuging, 10% w/v trichloroacetic acid solution (2.5 mL) was added to the mixture. After that, supernatant (2.5 mL) was mixed with distilled water (2.5 mL) and 0.1% (v/w) ferric chloride solution (0.5 mL). Finally, the samples were measured at the absorbance of 700 nm. 0.5 mL sample was replaced with 0.5 mL distilled water as blank reference. The higher the absorbance of final solution was, the stronger the reducing power. This experiment was performed in triplicate.
2.3.5. E-tongue analysis The taste of beverages was tested by a commercial e-tongue instrument (SA402B; Insent Inc., Atsugi-shi, Japan). Samples were diluted eight times to meet the measurement requirements. E-tongue had eight taste membranes, representing the tastes of sourness, bitterness, bitter aftertaste (aftertaste-A and aftertaste-B), astringency, umami, richness and saltiness. The process started from sensor check, and measurement started when five biofilm sensors (taste sensors) were stabilized. After one sample was measured, the sensors were cleaned and adjusted to measure the next sample. Each experiment was repeated four times.
2.4. Statistical analysis Microsoft Excel 2016 was used to obtain the mean and standard deviation and all data showed by mean ± standard deviation. Oneway analysis of variance (ANOVA) was applied in IBM SPSS 23.0 (IBM Inc., USA) to determine the significance level of difference between the mean values and 95% confidence level was used. Principal component analysis (PCA) is a method that can convert correlation variables into uncorrelated variables, which mainly uses the dimensionality reduction method to compress multivariate data (Granato et al., 2018). In the enose analysis, we performed PCA of e-nose sensor response values in the samples treated with different fermentation time or different enzyme ratios. PCA was also used in the taste response values of the e-tongue to
2.3.6. Sensory analysis Fermented C. militaris beverages were evaluated by a trained panel of 7 members (4 females and 3 males). Appearance, aroma, sweetness, sourness and consistency were analyzed. Each attribute counted for twenty points and the higher scores indicated the higher acceptance (Guo et al., 2018). 3
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
concentration of lactic acid bacteria was almost the same. The number of viable cells of yeast S. cerevisiae cannot be detected, because they had not been inoculated at this stage. As the 0Brix value, enzymatic hydrolysis group produced higher than that by control group, which may be related to the enzymatic process producing more reducing sugars (Table 1). After fermentation, the pH and 0Brix of all the samples dropped dramatically and the titratable acidity was on the sharp rise. Meanwhile, lactic acid bacteria increased nearly two orders of magnitude, but the growth of yeast S. cerevisiae was slow, which rose from 5 × 107 CFU/mL (initial concentration of yeast S. cerevisiae) to 8 × 107 CFU/mL. It can be speculated that lactic acid bacteria were the dominant species in this microecology, and yeast S.cerevisiae grew slowly probably because of the low pH. To be specific, lactic acid bacteria produce many organic acids after fermentation, leading to a decrease in the pH of fermentation broth, which may reduce the enzyme activity of yeast cells and inhibit its growth (Palmqvist & HahnHägerdal, 2000). Although there was no significant difference (P > 0.05) in the number of viable cells of yeast S. cerevisiae between control group and enzymatic hydrolysis group, the enumeration of lactic acid bacteria in the enzymatic hydrolysis group was significantly (P < 0.05) higher than that of the control group. The reason may that cellulase and pectinase destroyed the mycelial cell wall of C. militaris and released more reducing sugars and polysaccharides (Wang et al., 2014), which provided better nutrition environment for lactic acid bacteria growth. The cellulase/pectinase of 3:2 had the lowest pH value (3.44) and highest titratable acidity (10.82 g/L). It also contained the most lactic acid bacteria, although there was no significant difference (P < 0.05) compared with cellulase/pectinase of 2:3 and 1:1.
Table 1 Reducing sugars content of C. militaris pulp with different enzymatic hydrolysis. Cellulase (%)/Pectinase (%)
Reducing sugars (g Glu/L)
Control (without enzymatic hydrolysis) 1% cellulose alone only 1% pectinase alone
6.39 ± 0.04h 9.63 ± 0.10f 7.62 ± 0.17g
0.8/0.2 0.7/0.3 0.6/0.4 0.5/0.5 0.4/0.6 0.3/0.7 0.2/0.8
13.16 10.19 13.22 13.17 13.56 11.99 11.43
± ± ± ± ± ± ±
0.17b 0.15e 0.19b 0.03b 0.17a 0.16c 0.04d
One-way analysis of variance (n = 3) at 95% confidence level with different superscript letters (a, b, c, d, e, f, g and h) indicating a significant difference.
distinguish the flavors of the samples treated with different enzyme ratios. PCA data pretreatment was also carried out by SPSS 23.0. 3. Results and discussion 3.1. The reducing sugars content of Cordyceps militaris pulp Reducing sugars are a group of small molecule carbohydrates, which can be easily used by microorganism. As can be seen in Table 1, the complex enzymatic hydrolysis of cellulase and pectinase can significantly (P < 0.05) increase the content of reducing sugars in C. militaris pulp. Due to the synergistic effect of the two enzymes (Chong & Wong, 2015), the combined usage of enzymes was significantly (P < 0.05) better than that of using single enzyme. Different ratios of cellulase and pectinase can also affect the enzymatic hydrolysis (Sharma et al., 2017). When the amount of cellulase was 0.4% and pectinase was 0.6% (ratio of cellulase/pectinase = 2:3), the content of reducing sugars in the pulp was the highest (13.56 ± 0.17 g/L). It was more than twice as much as that of the non-enzymatic control group (6.39 ± 0.04 g/L). Cellulase/pectinase of 3:2 and 1:1 also contained high reducing sugars. Using the content of reducing sugars in C. militaris pulp to indicate the enzymatic hydrolysis effect, the cellulase/pectinase of 2:3 was found to be the best, followed by the cellulase/pectinase of 3:2 and 1:1. Subsequent experiments were carried out with these three ratios.
3.3. Flavor evaluation 3.3.1. E-nose analysis The effect of fermentation process and enzymatic hydrolysis on aroma was measured by an e-nose. In order to acquire the changes of aroma in different fermentation process, e-nose analysis was performed on C. militaris fermentation broth (without enzymatic hydrolysis) at different fermentation stages, namely 0 h, 12 h, 24 h, 36 h, 48 h and 60 h. The average response values of 14 sensors are shown in Fig. 2 (a). It is evident that the sample fermented for 60 h (after 48 h fermentation by lactic acid bacteria and 12 h fermentation by yeast S. cerevisiae) had the maximum response values. However, the response values of the samples fermented for 0–48 h (only fermented by lactic acid bacteria) were small. It means that lactic acid bacteria fermentation has little effect on the smell of the fermented beverages. What can be observed is that responding values of sensors increased remarkably for 60 h fermentation samples. Furthermore, the response values of S1, S5, S10, S13 sensors were all exceeded 2. Specific description of the gas sensor array in e-nose was mainly referred to the work reported by Chen, Zhang, Bhandari, and Guo (2018). It is said that S1, S5 and S13 sensors are sensitive to aromatic compounds (a class of gas components with
3.2. Changes in pH, titratable acidity, soluble solids, number of viable cells of lactic acid bacteria and yeast S. cerevisiae The pH, titratable acidity, soluble solids, number of viable cells of lactic acid bacteria and yeast S. cerevisiae of the unfermented substrate and fermented beverages can reflect the change of micro-ecological environment during fermentation. As shown in Table 2, before the fermentation, there was no significant difference (P > 0.05) in pH, titratable acidity, soluble solids among diverse samples. The initial
Table 2 pH, titratable acidity, oBrix, lactic acid bacteria and yeast cell count of unfermented substrate and fermented C. militaris beverage with different enzymatic hydrolysis. Treatments
Unfermented substrate
Fermented C. militaris beverage (after fermentation)
Control Cellulase/pectinase Cellulase/pectinase Cellulase/pectinase Control Cellulase/pectinase Cellulase/pectinase Cellulase/pectinase
pH
= 3:2 = 1:1 = 2:3 = 3:2 = 1:1 = 2:3
4.71 4.72 4.71 4.72 3.60 3.53 3.48 3.44
± ± ± ± ± ± ± ±
0.02a 0.01a 0.01a 0.01a 0.01A 0.01B 0.02C 0.01D
Titratable acidity (g/L)
0
2.82 ± 0.09a 2.94 ± 0.21a 3.13 ± 0.13a 3.01 ± 0.04a 10.60 ± 0.19A 10.66 ± 0.23A 10.74 ± 0.13A 10.82 ± 0.15A
16.65 16.85 17.15 17.05 15.55 15.85 15.65 15.70
Brix
± ± ± ± ± ± ± ±
0.05b 0.05ab 0.05a 0.15a 0.05A 0.05A 0.25A 0.10A
Lactic acid bacteria cell count (107 CFU/mL)
Yeast cell count (107 CFU/mL)
8.01 ± 0.12a 8.00 ± 0.09a 8.03 ± 0.07a 8.09 ± 0.11a 769.66 ± 24.32B 860.88 ± 60.15AB 926.11 ± 23.79A 934.15 ± 21.66A
N.D. N.D. N.D. N.D. 8.43 8.52 8.57 8.63
N.D., not detected. Different letters (a, b or A, B) in the same column indicate significant difference (p < 0.05). 4
± ± ± ±
0.60A 0.40A 0.50A 0.52A
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Fig. 3. Radar chart (a) and principal component analysis plot (b) about the changes of aroma of fermented C. militaris beverages obtained with different enzymatic hydrolysis (■: Control; ●: Cellulase/Pectinase 3:2; ▲: Cellulase/ Pectinase 1:1; ▼: Cellulase/Pectinase 2:3).
Fig. 2. Radar chart (a) and principal component analysis plot (b) about the changes of aroma of C. militaris fermentation broth during the whole fermentation process (■: 0 h; ●: 12 h; ▲: 24 h; ▼: 36 h; ♦: 48 h; ★: 48 + 12 h).
smaller odor difference among the enzymatic hydrolysis samples. Fermentation microorganisms of enzymatic hydrolysis group were more active than that of the control group (Table 2), which might produce more and richer volatile substances. Besides that, Urbonaviciene et al. pointed out the fermenting microorganisms can enhance the aroma of the fermented beverage (Urbonaviciene, Viskelis, Bartkiene, Juodeikiene, & Vidmantiene, 2015).
the pleasant scent). Moreover, S5 is sensitive to esters with the aromatic odor. It can be speculated that after two-step fermentation, esterification took place in the fermentation broth, which made the volatile flavor of the fermentation broth more prominent. And this is agreeable with Chan and Liu's (2019) research results. Fig. 2 (b) presents the principal component analysis (PCA) among the samples fermented at different time. There was little difference among the samples fermented for 0–48 h on the aroma, while the difference between the samples fermented for 60 h and that for 0–48 h was visible, which verified that the fermented C. militaris beverages would have more prominent volatile flavor after the subsequent yeast fermentation. All in all, two-step fermentation can make the beverage obtain a richer odor than before. Fig. 3 shows the effect of different enzymatic hydrolysis pretreatments on the volatile flavor components of fermented C. militaris beverages. As can be seen in Fig. 3 (a), compared with the control group, the enzymatic hydrolysis group had significantly higher response values on a series of sensors expect the S3, S4 and S14 sensors. The S1 response values with different ratios of enzymatic hydrolysis were all over 3 and the response value of S5 sensor with cellulase/pectinase of 1:1 was also above 3, which indicated that the enzymatic hydrolysis pretreatment could make the fermented beverage to have a rich aroma. The PCA presented in Fig. 3(b) more clearly shows that there was a significant difference (P < 0.05) in aroma between the control group and the enzymatic hydrolysis group, and the discrimination index (DI) of PCA was 90.8%. Moreover, the location of each enzymatic hydrolysis sample was much closer when compared with the control, which implied
3.3.2. E-tongue analysis E-tongue can convert electrical signals into taste signals to distinguish the taste of the fermented beverages, which has a small threshold of sensation and can well exclude the subjectivity of sensory evaluation (Jiang, Zhang, Bhandari, & Adhikari, 2018). Fig. 4 (a) displays the taste changes of C. militaris fermentation broth without enzymatic hydrolysis for 0 h, 12 h, 24 h, 36 h, 48 h and 60 h. It can be seen from the figure that with the fermentation time prolonging, the sourness of the fermentation broth gradually increased, the bitterness gradually decreased, while the signal values of the remaining taste sensors did not change obviously. The reduction of bitterness may be due to the formation of large amounts of acids masking other tastes. Furthermore, yeast fermentation produced small amount of alcohol, providing a mellow sense of the beverage (Lorenzini, Simonato, Slaghenaufi, Ugliano, & Zapparoli, 2019). Fig. 4 (b) represents the effect of different enzymatic hydrolysis pretreatments on the taste of the C. militaris beverages after two-step fermentation. As is shown in the picture that the enzymatic hydrolysis treatments had some effects on the sourness, 5
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Fig. 5. The principal component analysis (PCA) plot about the changes of taste of fermented C. militaris beverages obtained with different enzymatic hydrolysis ( : Control; ■: Cellulase/Pectinase 3:2; ▲: Cellulase/Pectinase 1:1; ★: Cellulase/Pectinase2:3).
3.3.3. Sensory analysis As can be seen in Table 3, the total score of the enzymatic hydrolysis group was significantly higher than that of control group, and all the scores of enzymatic hydrolysis group exceeded 80 points. The sensory scores for appearance were similar among the samples. The aroma scores were higher in the enzymatic group than that in the control group, and the highest scores were obtained in the cellulase/pectinase of 1:1. In the previous e-nose analysis, the cellulase/pectinase of 1:1 had the moderate aroma response values (Fig. 3 (a)). Sweetness and sourness may be related to the 0Bix, pH value and soluble solids. Concerning the consistency of beverages, the difference is not significant, which means the beverages are all uniform and stable. All in all, compared to the control group, the flavor of the enzymatic group was improved (see Table 4).
3.4. Nutritional quality analysis Fig. 4. Radar charts performed with e-tongue date. (a) Fermentation broth with different fermentation time (■: 0 h; ●: 12 h; ▲: 24 h; ▼: 36 h; ♦: 48 h; ★: 48 + 12 h); (b) Fermented beverages completed two-step fermentation with different enzymatic hydrolysis treatments (■: Control; ●: Cellulase/Pectinase 3:2; ▲: Cellulase/Pectinase 1:1; ▼: Cellulase/Pectinase 2:3).
3.4.1. Cordycepin The content of cordycepin in the fermented C. militaris beverage can reflect its nutritional quality (Jin et al., 2018). We could conclude from Fig. 6 (a) that the content of cordycepin increased significantly (P < 0.05) after enzymatic hydrolysis treatments, while there was no significant difference (P > 0.05) among different ratios of enzymes. Although the fermentation process would reduce the cordycepin, the group of cellulase/pectinase with the ratio of 2:3 had excellent retention (as high as 98.02%) of cordycepin. Regarding the group of cellulase/pectinase with the ratio of 3:2, it had the high content of cordycepin in the unfermented substrate with low retention of cordycepin after fermentation, but the content was still higher than that of the control group. From the above analysis, the enzymatic group with the cellulase/pectinase of 1:1 and 2:3, both on the quantity and retention of cordycepin were significantly better (P < 0.05) than that of the control group. Overall, enzymatic treatments had a good effect on cordycepin retention of C. militaris fermented beverage. According to the above analysis, there were more nutrients available to the microorganisms in the fermentation broth after enzymatic hydrolysis, which may protect the cordycepin from decomposition.
bitterness and astringency of the fermented beverages. The maximum difference in response values of sourness, bitterness and astringency between the control and enzymatic group was 0.4, 0.14 and 0.84, respectively. The most dominant difference was the astringency value of 3:2 group bigger than other samples. Fig. 5 shows the principal component analysis of fermented beverages between the control group and enzymatic hydrolysis group. According to Fig. 5, samples with cellulase/pectinase of 1:1 and 2:3 were close to the control group. In addition, the sample with cellulase/ pectinase of 3:2 separated from the control group obviously, which implicated the taste of these two samples were the most different. In general, the data points are scattered and interactive. It represents that the enzymatic hydrolysis has little effect on the taste of the fermented drinks.
6
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Table 3 Sensory analysis results of samples prepared by different enzymatic hydrolysis treatments. Treatments Control Cellulase/pectinase = 3:2 Cellulase/pectinase = 1:1 Cellulase/pectinase = 2:3
Appearance 16.14 16.41 16.91 15.91
± ± ± ±
Aroma b
0.58 0.39b 0.30a 0.33b
14.51 17.41 18.11 16.81
Sweetness ± ± ± ±
c
1.15 0.42ab 0.42a 0.70b
15.20 16.10 15.25 17.51
± ± ± ±
Sourness c
0.80 0.37b 0.85c 0.64a
16.11 15.30 16.21 15.94
± ± ± ±
Consistency a
0.74 0.32b 0.56a 0.49ab
14.90 15.51 15.61 16.37
± ± ± ±
Total scores b
1.30 0.62ab 0.66ab 1.60a
76.87 80.74 82.11 82.56
± ± ± ±
1.75c 1.22b 1.07ab 0.89a
Different letters (a, b, c) of the same column indicate significant differences (P < 0.05). Table 4 Retention of cordycepin in the fermented C. militaris beverages with different enzymatic hydrolysis treatments. Cellulase/Pectinase
Control
3:2
1:1
2:3
Retention of cordycepin (%)
88.84 ± 5.11c
79.94 ± 2.96d
90.38 ± 2.89b
98.02 ± 0.36a
One-way analysis of variance (n = 3) at 95% confidence level with different superscript letters (a, b, c, d) indicating a significant difference.
Fig. 6. Nutritional quality of the fermented C. militaris beverages: (a) Changes of cordycepin in unfermented and fermented C. militaris beverages with different : Unfermented substrate; : Fermented beverages), (b) hydroxyl radical scavenging rate % ( ) and (c) reducing power enzymatic hydrolysis treatments ( ) of the two-step fermented beverages with different enzymatic hydrolysis treatments. (A700nm: 7
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
3.4.2. Antioxidant activity Antioxidant activity is also an important quality parameter of the fermented beverage. The antioxidant capacity of fermented C. militaris beverage was evaluated by the hydroxyl radical scavenging rate (%) and reducing power (A700nm) of the fermentation broth. From the results presented in Fig. 6 (b) and Fig. 6 (c), it can be found that the enzymatic hydrolysis pretreatment can significantly (P < 0.05) improve the antioxidant capacity of fermented C. militaris beverage. When compared with the control, enzymatic hydrolysis group with the cellulase/pectinase of 3:2, 1:1 and 2:3 increased the hydroxyl radical scavenging rate by 14.20%, 16.08% and 28.76%, and improved the reducing power by 3.67%, 2.06% and 6.91%, respectively. This is presumably because enzymatic hydrolysis treatments increased the content of reducing sugars and cordycepin in the fermentation broth, which made the fermentation broth achieve more powerful antioxidant capacity. The antioxidant analysis is similar to the previously reported by Wu, Zhang, and Bhandari (2019).
Blandino, A., Al-Aseeri, M. E., Pandiella, S. S., Cantero, D., & Webb, C. (2003). Cerealbased fermented foods and beverages. Food Research International, 36(6), 527–543. Cai, L., Xiong, X., Lu, Z., & Sun, X. (2012). Purification and mutagenesis of Glucoamylase production strain and making technology of composite Koji. Food and Fermentation Technology, 48(6), 20–23. Champagne, C. P., Gomes da, C. A., & Daga, M. (2018). Strategies to improve the functionality of probiotics in supplements and foods. Current Opinion in Food Science, 22, 160–166. Chan, W., & Liu, S. (2019). The effects of carbohydrase, probiotic Lactobacillus paracasei and yeast Lindnera saturnus on the composition of a novel okara (soybean residue) functional beverage. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 100, 196–204. Chen, H. Z., Zhang, M., Bhandari, B., & Guo, Z. (2018). Evaluation of the freshness of fresh-cut green bell pepper (Capsicum annuum var. grossum) using electronic nose. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 87, 77–84. Chong, S. Y., & Wong, C. W. (2015). Production of spray-dried Sapodilla (Manilkara zapota) powder from enzyme-aided liquefied puree. Journal of Food Processing and Preservation, 39(6), 2604–2611. Das, S. K., Masuda, M., Sakurai, A., & Sakakibara, M. (2010). Medicinal uses of the mushroom Cordyceps militaris: Current state and prospects. Fitoterapia, 81(8), 961–968. Feng, Y., Zhang, M., Mujumdar, A. S., & Gao, Z. (2017). Recent research process of fermented plant extract: A review. Trends in Food Science & Technology, 65, 40–48. Granato, D., Putnik, P., Kovačević, D. B., et al. (2018). Trends in chemometrics: Food authentication, microbiology, and effects of processing. Comprehensive Reviews in Food Science and Food Safety, 17(3), 663–677. Guo, J., Yan, Y., Wang, M., Wu, Y., Liu, S. Q., Chen, D., et al. (2018). Effects of enzymatic hydrolysis on the chemical constituents in jujube alcoholic beverage fermented with Torulaspora delbrueckii. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 97(May), 617–623. Hancioǧlu, Ö., & Karapinar, M. (1997). Microflora of Boza, a traditional fermented Turkish beverage. International Journal of Food Microbiology, 35(3), 271–274. Jiang, H., Zhang, M., Bhandari, B., & Adhikari, B. (2018). Application of electronic tongue for fresh foods quality evaluation: A review. Food Reviews International, 34(8), 746–769. Jin, Y., Meng, X., Qiu, Z., Su, Y., Yu, P., & Qu, P. (2018). Anti-tumor and anti-metastatic roles of cordycepin, one bioactive compound of Cordyceps militaris. Saudi Journal of Biological Sciences, 25(5), 991–995. Lim, J. Y., Yoon, H. S., Kim, K. Y., Kim, K. S., Noh, J. G., & Song, I. G. (2010). Optimum conditions for the enzymatic hydrolysis of citron waste juice using response surface methodology (RSM). Food Science and Biotechnology, 19(5), 1135–1142. Lorenzini, M., Simonato, B., Slaghenaufi, D., Ugliano, M., & Zapparoli, G. (2019). Assessment of yeasts for apple juice fermentation and production of cider volatile compounds. Lwt- Food Science and Technology, 99, 224–230. Marsh, A. J., Hill, C., Ross, R. P., & Cotter, P. D. (2014). Fermented beverages with healthpromoting potential: Past and future perspectives. Trends in Food Science & Technology, 38, 113–124. Masuda, M., Urabe, E., Sakural, A., & Sakakibara, M. (2006). Production of cordycepin by surface culture using the medicinal mushroom Cordyceps militaris. Enzyme and Microbial Technology, 39, 641–646. Ma, L., Zhang, M., Bhandari, B., & Gao, Z. (2017). Recent developments in novel shelf life extension technologies of fresh-cut fruits and vegetables. Trends in Food Science and Technology, 64, 23–38. Oyaizu, M. (1986). Antioxidant activity of browning products of glucosamine fractionated by organic solvent and thin-layer chromatography. Nippon Shokulin Kogyo Gakkaishi, 35, 771–775. Palmqvist, E., & Hahn-Hägerdal, B. (2000). Fermentation of lignocellulosic hydrolysates. II: Inhibitors and mechanisms of inhibition. Bioresource Technology, 74(1), 25–33. Phan, C. W., David, P., Tan, Y. S., Naidu, M., Wong, K. H., Kuppusamy, U. R., et al. (2014). Intrastrain comparison of the chemical composition and antioxidant activity of an edible mushroom, pleurotus giganteus, and its potent neuritogenic properties. Scientific World Journal, 1–10 2014. Phan, C. W., Wang, J. K., Cheah, S. C., Naidu, M., David, P., & Sabaratnam, V. (2018). A review on the nucleic acid constituents in mushrooms: Nucleobases, nucleosides and nucleotides. Critical Reviews in Biotechnology, 38(5), 762–777. Sharma, H. P., Patel, H., & Sugandha (2017). Enzymatic added extraction and clarification of fruit juices: A review. Critical Reviews in Food Science and Nutrition, 57(6), 1215–1227. Smirnoff, N., & Cumbes, Q. J. (1989). Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry, 28(4), 1057–1060. Urbonaviciene, D., Viskelis, P., Bartkiene, E., Juodeikiene, G., & Vidmantiene, D. (2015). The use of lactic acid bacteria in the fermentation of fruits and vegetables - technological and functional properties. Biotechnology, 135–164 April. Wang, Z., Wang, R., Wu, Y., Zhou, F., Hua, C., Gu, Z., et al. (2014). Optimization of the extraction Cordyceps militaris polysaccharides by microwave assisted with enzyme hydrolysis. Science and Technology of Food Industry, 35(11), 188–192. Woraharn, S., Lailerd, N., Sivamaruthi, B. S., Wangcharoen, W., Sirisattha, S., Peerajan, S., et al. (2016). Evaluation of factors that influence the L-glutamic and γ-aminobutyric acid production during Hericium erinaceus fermentation by lactic acid bacteria. CyTA - Journal of Food, 14(1), 47–54. Wu, X., Zhang, M., & Bhandari, B. (2019). A novel Infrared Freeze drying ( IRFD ) technology to lower the energy consumption and keep the quality of cordyceps militaris. Innovative Food Science & Emerging Technologies, 54, 34–42. Yang, R., Gu, D., & Gu, Z. (2016). Cordyceps rice wine: A novel brewing process. Journal of Food Process Engineering, 39(6), 581–590. Zhang, M., Chen, H., Mujumdar, A. S., Tang, J., Miao, S., & Wang, Y. (2017). Recent
4. Conclusions A nutrient-rich fermented C. militaris beverage that is dark reddishbrown, clear and transparent, sweet and sour was obtained. Enzymatic hydrolysis can produce more reducing sugars, which is conducive to the subsequent fermentation process. Two-step fermentation with lactic acid bacteria and yeasts yielded a pleasant fragrant and aromatic odor, making the fermented beverage acquire a pleasant taste. Besides, enzymatic hydrolysis treatments could enhance the volatile flavor profile of fermented beverages. With the use of cellulase/pectinase ratio of 2:3, cordycepin was extracted more in the unfermented substrate and had a better retention after fermentation. The enzymatic treatments also significantly improved the antioxidant activity of the fermented C. militaris beverage. Overall, enzymatic hydrolysis pretreatment with fermentation provided a promising method for improving the flavor and enhancing the nutritional quality of fermented beverage. The shelflife of this functional fermented beverage will be the subject of future study. Author contributions Literature research and experiments were performed by Yanyan Lao. Oversight of the project and field experience were provided by Min Zhang, Zhongqin Li, and Bhesh Bhandari. Critical review of the manuscript was performed by Min Zhang and Bhesh Bhandari. Declaration of competing interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript. Acknowledgments We acknowledge the financial support from the National Key R&D Program of China (Contract No. 2018YFD0400801), the 111 Project (BP0719028), the Yangzhou City Agricultural Key R&D Program (No.YZ2019034), Jiangsu Province (China) “Collaborative Innovation Center for Food Safety and Quality Control” Industry Development Program and the National First-class Discipline Program of Food Science and Technology (No. JUFSTR20180205), all of which enabled us to carry out this study. References Altay, F., Karbancioglu-Güler, F., Daskaya-Dikmen, C., & Heperkan, D. (2013). A review on traditional Turkish fermented non-alcoholic beverages: Microbiota, fermentation process and quality characteristics. International Journal of Food Microbiology, 167(1), 44–56.
8
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Zhang, M., Li, C. L., & Ding, X. L. (2005). Effects of heating conditions on the thermal denaturation of white mushroom suitable for dehydration. Drying Technology, 23(5), 1119–1125. Zhang, M., Zhang, C. J., & Shrestha, S. (2005). Study on the preparation technology of superfine ground powder of Agrocybe chaxingu Huang. Journal of Food Engineering, 67(3), 333–337.
developments in high-quality drying of vegetables, fruits and aquatic products. Critical Reviews in Food Science and Nutrition, 57(6), 1239–1255. Zhang, Y., Fraatz, M. A., Birk, F., Rigling, M., Hammer, A., & Zorn, H. (2018). Enantiomeric ratios of 2-methylbutanoic acid and its methyl ester: Elucidation of novel biogenetic pathways towards (R)-methyl 2-methylbutanoate in a beverage fermented with shiitake. Food Chemistry, 266(June), 475–482.
9
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
A novel combination of enzymatic hydrolysis and fermentation: Effects on the flavor and nutritional quality of fermented Cordyceps militaris beverage
T
Yanyan Laoa,b, Min Zhanga,c,∗, Zhongqin Lid,e, Bhesh Bhandarif a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, Jiangsu, China c Jiangsu Province Key Laboratory of Advanced Food Manufacturing Equipment and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China d Yandi Biological Engineering Co., Ltd, Changde, Hunan, China e Yangzhou Yechun Food Production & Distribution Co, Yangzhou, 225200, Jiangsu, China f School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Enzymatic hydrolysis Two-step fermentation Flavor Nutritional quality Fermented Cordyceps militaris beverage
This study evaluated the effects of enzymatic hydrolysis and fermentation on the flavor and nutritional quality of fermented Cordyceps militaris beverage. Enzymatic hydrolysis can produce more reducing sugars and provide an appropriate fermentation substrate for the growth of bacteria. Electronic nose (e-nose) analysis indicated that first enzymatic hydrolysis and then two-step fermentation could significantly increase the volatile flavor component response values of the fermented beverage. Moreover, electronic tongue (e-tongue) analysis indicated that the fermentation process can increase the sourness and decrease the bitterness of the beverage, resulting in attaining a better taste. Based on the research undertaken on nutritional quality of this fermentation beverage, it was found that a combination of cellulase and pectinase with the ratio of 2:3 yielded higher cordycepin content. After fermentation, the retention rate of cordycepin was also high (98.02%). Fermented beverage pretreated with complex enzymes had a stronger antioxidant capacity compared with non-enzymatic group, which was indicated by the hydroxyl radical scavenging rate and reducing power determination. This fermented C. militaris beverage is expected to help to develop a functional beverage and further research can be carried out on the shelf-life of this beverage.
1. Introduction Cordyceps militaris, food-grade macrofungi belonging to the Ascomycete class, has been widely used as folk tonic food or crude drug in East Asia (Das, Masuda, Sakurai, & Sakakibara, 2010). It is rich in protein, amino acids, and contains diverse biologically active substances such as cordyceps polysaccharides cordycepin, cordycepic acid (D-mannitol) and superoxide dismutase (SOD) (Phan et al., 2018). The main nutrient substance of C. militaris is cordycepin, having various physiological functions such as anti-tumor, hypoglycemic, immunomodulatory, antibacterial and anti-inflammatory (Yang, Gu, & Gu, 2016). It is said that the medicinal value of C. militaris is similar to Cordyceps sinensis and C. militaris are known for their high content of cordycepin (Masuda, Urabe, Sakural, & Sakakibara, 2006). Fresh C. militaris is easy to spoil and can be dried to extend its shelf-life (Zhang et al., 2017). Besides drying, it can also be fermented to delay spoilage (Ma, Zhang, Bhandari, & Gao, 2017; Marsh, Hill, Ross, & Cotter, 2014). Fermented plant extract (FPE) is a new type of fermented beverage ∗
that originated in Japan. FPE is generally made from vegetables, fruits, cereals, legumes, edible fungi (although not plant), which contains abundant nutrients such as enzymes, vitamins, minerals, esters and polyphenols (Feng, Zhang, Mujumdar, & Gao, 2017). FPE is usually fermented by various probiotics, such as lactic acid bacteria, acetic acid bacteria and yeasts (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003). Probiotics are increasingly being used in food products due to their excellent biological activities (Champagne, Gomes da, & Daga, 2018). FPE is different from other fermented products such as wine and yogurt because it contains no alcohol or tiny amount of it (Altay, Karbancioglu-Güler, Daskaya-Dikmen, & Heperkan, 2013), and is not a dairy based food. Edible fungi have high nutrition value of which the content of protein and amino acids, fibers, vitamins, minerals are high, and the fat content is low (Phan et al., 2014; Zhang, Li, & Ding, 2005; Zhang, Zhang, & Shrestha, 2005). There are some research works reported in relation to edible fungi beverage. Hericium erinaceus beverage was found rich in L-glutamic and γ-aminobutyric acid (Woraharn et al., 2016), and shiitake beverage contained important flavor compound
Corresponding author. School of Food Science and Technology, Jiangnan University, 214122 Wuxi, Jiangsu Province, China. E-mail address: [email protected] (M. Zhang).
https://doi.org/10.1016/j.lwt.2019.108934 Received 5 October 2019; Received in revised form 5 December 2019; Accepted 5 December 2019 Available online 07 December 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
preparation process is presented as follows. All treatments were conducted in triplicate. C. militaris pulp preparation: After the C. militaris powder and distilled water mixed in the ratio of 1:10, C. militaris pulp was obtained by intermittent stirring for 2 min (each duration of 10 s) with a high-speed homogenizer (T18BS25; IKA Co., Ltd., Germany) at the rotating speed of 3000–3600 r/min. Enzymatic hydrolysis of C. militaris pulp: Firstly, the pH of C. militaris pulp was adjusted to 4.80 ± 0.05 with 10% w/v food-grade citric acid solution. Then, different ratios of cellulase (0.2%–0.8%, w/v, 50 kU/g, Imperial Jade Biotechnology Co., Ltd., Ningxia, China) and pectinase (0.2%–0.8%, w/v, 100 kU/g, Ruiming Food Additive Co., Ltd., Henan, China) were added. The amount of enzyme should be minimal as it may result in higher production costs (Chong & Wong, 2015). Based on pre-test, the total amount of 1% w/v enzyme was enough. Enzymatic hydrolysis was carried out in a water bath at 50 °C for 4 h. After enzymatic hydrolysis, the enzymes were inactivated at 90 °C for 5 min. Fermentation substrate preparation: 30% w/v C. militaris pulp (with or without enzymatic hydrolysis), 15% w/v brown sugar (Ganzhiyuan sugar industry Co., Ltd., Nanjing, China) and 55% w/v distilled water were mixed uniformly and pasteurized at 85 °C for 10 min. The fermented substrate was cooled to 42 °C and poured into pre-sterilized glass fermenters. Fermentation: The initial pH of the fermentation substrate was adjusted to 4.60–5.00. During the initial phase, the fermentation substrate was inoculated with 0.1% w/v lactic acid bacteria powder (Baishengyou Biotechnology Co., Ltd., Suzhou, China) that contained five strains (Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium lactis and Bifidobacterium longum). The lactic acid fermentation was done at 28 °C for 48 h followed by inoculating 0.1% w/v yeast (Saccharomyces cerevisiae; Angel Yeast Co., Ltd., Hubei, China) to ferment at 28 °C for 12 h. The initial concentration of lactic acid bacteria and yeast S. cerevisiae was 8 × 107 colony-forming units (CFU)/ mL and 5 × 107 CFU/mL, respectively. Fermentation was carried out under anaerobic conditions. After fermentation, the suspension was centrifuged (4200 r/min,
(R)-methyl 2-methyl butanoate up to 35% (Zhang et al., 2018). However, there are only few reports on developing fermented C. militaris beverage. It may be due to the high fiber content of C. millitaris (Phan et al., 2014), which probably affects the fermentation process (Guo et al., 2018). Furthermore, C. militaris has a characteristic odor which is not an acceptable flavor for beverage and needs to be removed or masked by processing. Enzymatic hydrolysis can degrade macromolecules into small molecules (such as low molecular weight sugars) (Sharma, Patel, & Sugandha, 2017). Cellulase and pectinase with the synergistic effect can loosen the structure of materials efficiently due to breakdown of cellulose and pectin into smaller molecules (Lim et al., 2010). This function may hopefully make the water-soluble nutrients (cordycepin) extracted out easily. Besides, the fermentation with probiotics not only can improve the sensory quality but also promote the bioavailability of nutrients (Hancioǧlu & Karapinar, 1997). According to the available literature, the research on the effects of enzymatic hydrolysis and fermentation on C. militaris fermented beverage is rare. Therefore, the purpose of this research work is to study the effects of enzymatic hydrolysis (cellulase and pectinase) and fermentation on pH, titratable acidity, soluble solids (0Brix) and fermenting microorganisms. Moreover, flavor evaluation (e-nose, e-tongue, sensory analysis) and nutritional quality analysis (cordycepin content and antioxidant activity) of C. militaris fermented beverage were carried out, striving to set this fermentation process as a reference for the same category of fermented beverages. 2. Materials and methods 2.1. Materials Dried C. militaris fungi provided by Yandi Biological Engineering Co., Ltd., Hunan, China were smashed through a 100-mesh sieve to obtain the powder and stored in a desiccator before experimentation. 2.2. C. militaris pulp preparation, enzymatic hydrolysis and fermentation The process flow chart is summarized in Fig. 1 and specific
Fig. 1. Flow chart of fermented C. militaris beverage. 2
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
10 min), filtered and sterilized (boiling water bath, 5 min) to obtain the fermented C. militaris beverage. Control: The treatment conditions were the same except that there was no enzymatic hydrolysis treatment of C. militaris pulp.
2.3.7. Measurement of cordycepin content The determination of cordycepin content was according to the protocol of Agricultural Industry Standard of the People's Republic of China (NY/T 2116-2012). Unfermented substrate or fermented beverage (including the experimental group and the control group) was accurately weighed (5 g) and poured into 50 mL volumetric flasks, and then added with about 40 mL of distilled water. After sonication for 30 min, the samples were adjusted to 50 mL with distilled water. Each sample was passed through the 0.45 μm microfiltration membrane. The filtrates were measured by high-performance liquid chromatography (HPLC). Cordycepin content of unfermented substrate (C0) and fermented beverages (C1) were obtained. The retention of cordycepin (%) was calculated as follows:
2.3. Analytical methods 2.3.1. Measurement of the reducing sugars content in C. militaris pulp The content of reducing sugars in C. militaris pulp was determined by 3, 5-dinitrosalicylic acid (DNS) colorimetric method, (Cai, Xiong, Lu, & Sun, 2012). C. militaris pulp was centrifuged (5400 r/min, 20 min) and filtered. The filtrate (1 mL) was mixed with DNS (2 mL) and incubated in the boiling water for 2 min. Then, the mixture was cooled and made up to 25 mL with distilled water. The absorbance was measured at 540 nm. Reducing sugars expressed as grams of equivalent Glu per liter of every sample (g Glu/L). The experiment was performed in triplicate.
Retention of cordycepin (%) = C0/C1 Thermo Fisher Scientific Ultimate3000 system (Thermo Fisher Scientific, Co., Ltd., USA) and a C18 reverse-phase column (250 mm × 4.6 mm i.d., 5 μm) were used to measure the content of cordycepin. The mobile phase consisted of 5% acetonitrile and 95% ultrapure water with a flow rate of 1.0 mL/min. Detection wavelength was set as 260 nm and column temperature was 35 °C. The injection volume of each sample was 10 μL and the detection time was 22 min. The experiment was performed in duplicate.
2.3.2. Measurement of the pH, titratable acidity and soluble solids (0Brix) The pH value was directly measured by a pH meter (Starter 3100; Ohaus instrument Co., Ltd., Shanghai, China) at room temperature. The content of titratable acidity was determined by acid-base titration method following the National Standard of the People's Republic of China (GB/T 12456-2008). A refractometer (WZS-12W) was used to measure soluble solid (0Brix) at 20 °C. The experiment was performed in triplicate.
2.3.8. Hydroxyl radical scavenging rate and reducing power Hydroxyl radical scavenging rate was determined by the salicylic acid method (Smirnoff & Cumbes, 1989). The reaction system contained 1 mL 8.8 mmol/L hydrogen peroxide, 1 mL 9 mmol/L ferrous sulfate, 1 mL 9 mmol/L salicylic acid-ethanol solution, 0.5 mL different treatments fermented beverages with the total volume of the reactants 15 mL (the insufficient volume was made up with distilled water). The final mixture was incubated at 37 °C for 30 min. The absorbance (Ax) was measured with the UV2600 spectrophotometer (Tianmei Scientific Instrument Co., Ltd., Shanghai, China) at 510 nm. Blank control group and another control group were measured as the background absorption of reagent (Ao) and sample (Axo), respectively. The experiment was performed in triplicate.
2.3.3. Enumeration the viable cells of lactic acid bacteria and yeast The viable cells concentration of lactic acid bacteria and yeast S. cerevisiae was determined by plate count method according to the standard of GB 4789.35-2016 and GB 4789.15-2016. All samples were serial tenfold diluted with 0.85% saline solution and were used to inoculate in MRS agar plates for the enumeration of the Lactic acid bacteria and potato dextrose agar plates for the enumeration of the yeast S. cerevisiae. The experiment was performed in duplicate. 2.3.4. E-nose analysis E-nose (iNose; Ruifen Trading Co., Ltd, Shanghai, China) was used to distinguish volatile flavor components of fermented beverages. C. militaris fermentation broth (4 g) was added to the 40 mL sealed vials and left standing at constant temperature (28 °C) for 30 min. The enose, equipped with 14 metal oxide semiconductor gas sensors, has different response values to different smells. Before experiment, the enose needs to be cleaned for 3000 s to ensure the accuracy of subsequent measurement. Measurement stage lasted for 120 s, and gas was delivered to the sensor chamber at the flow rate of 1 L/min. Recovery time (cleaning time) for sensors was 150 s. The experiment was repeated four times.
Hydroxyl radical scavenging rate = [1-(Ax-Axo)/Ao] × 100% A slightly modified method of Oyaizu (1986) was used to analysis the reducing power. Different samples (0.5 mL) were mixed with pH 6.6 phosphate buffer solution (2.5 mL) and 1% w/v potassium ferricyanide solution (2.5 mL). The mixture was heated in a 50 °C water bath for 20 min. After rapidly cooling and centrifuging, 10% w/v trichloroacetic acid solution (2.5 mL) was added to the mixture. After that, supernatant (2.5 mL) was mixed with distilled water (2.5 mL) and 0.1% (v/w) ferric chloride solution (0.5 mL). Finally, the samples were measured at the absorbance of 700 nm. 0.5 mL sample was replaced with 0.5 mL distilled water as blank reference. The higher the absorbance of final solution was, the stronger the reducing power. This experiment was performed in triplicate.
2.3.5. E-tongue analysis The taste of beverages was tested by a commercial e-tongue instrument (SA402B; Insent Inc., Atsugi-shi, Japan). Samples were diluted eight times to meet the measurement requirements. E-tongue had eight taste membranes, representing the tastes of sourness, bitterness, bitter aftertaste (aftertaste-A and aftertaste-B), astringency, umami, richness and saltiness. The process started from sensor check, and measurement started when five biofilm sensors (taste sensors) were stabilized. After one sample was measured, the sensors were cleaned and adjusted to measure the next sample. Each experiment was repeated four times.
2.4. Statistical analysis Microsoft Excel 2016 was used to obtain the mean and standard deviation and all data showed by mean ± standard deviation. Oneway analysis of variance (ANOVA) was applied in IBM SPSS 23.0 (IBM Inc., USA) to determine the significance level of difference between the mean values and 95% confidence level was used. Principal component analysis (PCA) is a method that can convert correlation variables into uncorrelated variables, which mainly uses the dimensionality reduction method to compress multivariate data (Granato et al., 2018). In the enose analysis, we performed PCA of e-nose sensor response values in the samples treated with different fermentation time or different enzyme ratios. PCA was also used in the taste response values of the e-tongue to
2.3.6. Sensory analysis Fermented C. militaris beverages were evaluated by a trained panel of 7 members (4 females and 3 males). Appearance, aroma, sweetness, sourness and consistency were analyzed. Each attribute counted for twenty points and the higher scores indicated the higher acceptance (Guo et al., 2018). 3
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
concentration of lactic acid bacteria was almost the same. The number of viable cells of yeast S. cerevisiae cannot be detected, because they had not been inoculated at this stage. As the 0Brix value, enzymatic hydrolysis group produced higher than that by control group, which may be related to the enzymatic process producing more reducing sugars (Table 1). After fermentation, the pH and 0Brix of all the samples dropped dramatically and the titratable acidity was on the sharp rise. Meanwhile, lactic acid bacteria increased nearly two orders of magnitude, but the growth of yeast S. cerevisiae was slow, which rose from 5 × 107 CFU/mL (initial concentration of yeast S. cerevisiae) to 8 × 107 CFU/mL. It can be speculated that lactic acid bacteria were the dominant species in this microecology, and yeast S.cerevisiae grew slowly probably because of the low pH. To be specific, lactic acid bacteria produce many organic acids after fermentation, leading to a decrease in the pH of fermentation broth, which may reduce the enzyme activity of yeast cells and inhibit its growth (Palmqvist & HahnHägerdal, 2000). Although there was no significant difference (P > 0.05) in the number of viable cells of yeast S. cerevisiae between control group and enzymatic hydrolysis group, the enumeration of lactic acid bacteria in the enzymatic hydrolysis group was significantly (P < 0.05) higher than that of the control group. The reason may that cellulase and pectinase destroyed the mycelial cell wall of C. militaris and released more reducing sugars and polysaccharides (Wang et al., 2014), which provided better nutrition environment for lactic acid bacteria growth. The cellulase/pectinase of 3:2 had the lowest pH value (3.44) and highest titratable acidity (10.82 g/L). It also contained the most lactic acid bacteria, although there was no significant difference (P < 0.05) compared with cellulase/pectinase of 2:3 and 1:1.
Table 1 Reducing sugars content of C. militaris pulp with different enzymatic hydrolysis. Cellulase (%)/Pectinase (%)
Reducing sugars (g Glu/L)
Control (without enzymatic hydrolysis) 1% cellulose alone only 1% pectinase alone
6.39 ± 0.04h 9.63 ± 0.10f 7.62 ± 0.17g
0.8/0.2 0.7/0.3 0.6/0.4 0.5/0.5 0.4/0.6 0.3/0.7 0.2/0.8
13.16 10.19 13.22 13.17 13.56 11.99 11.43
± ± ± ± ± ± ±
0.17b 0.15e 0.19b 0.03b 0.17a 0.16c 0.04d
One-way analysis of variance (n = 3) at 95% confidence level with different superscript letters (a, b, c, d, e, f, g and h) indicating a significant difference.
distinguish the flavors of the samples treated with different enzyme ratios. PCA data pretreatment was also carried out by SPSS 23.0. 3. Results and discussion 3.1. The reducing sugars content of Cordyceps militaris pulp Reducing sugars are a group of small molecule carbohydrates, which can be easily used by microorganism. As can be seen in Table 1, the complex enzymatic hydrolysis of cellulase and pectinase can significantly (P < 0.05) increase the content of reducing sugars in C. militaris pulp. Due to the synergistic effect of the two enzymes (Chong & Wong, 2015), the combined usage of enzymes was significantly (P < 0.05) better than that of using single enzyme. Different ratios of cellulase and pectinase can also affect the enzymatic hydrolysis (Sharma et al., 2017). When the amount of cellulase was 0.4% and pectinase was 0.6% (ratio of cellulase/pectinase = 2:3), the content of reducing sugars in the pulp was the highest (13.56 ± 0.17 g/L). It was more than twice as much as that of the non-enzymatic control group (6.39 ± 0.04 g/L). Cellulase/pectinase of 3:2 and 1:1 also contained high reducing sugars. Using the content of reducing sugars in C. militaris pulp to indicate the enzymatic hydrolysis effect, the cellulase/pectinase of 2:3 was found to be the best, followed by the cellulase/pectinase of 3:2 and 1:1. Subsequent experiments were carried out with these three ratios.
3.3. Flavor evaluation 3.3.1. E-nose analysis The effect of fermentation process and enzymatic hydrolysis on aroma was measured by an e-nose. In order to acquire the changes of aroma in different fermentation process, e-nose analysis was performed on C. militaris fermentation broth (without enzymatic hydrolysis) at different fermentation stages, namely 0 h, 12 h, 24 h, 36 h, 48 h and 60 h. The average response values of 14 sensors are shown in Fig. 2 (a). It is evident that the sample fermented for 60 h (after 48 h fermentation by lactic acid bacteria and 12 h fermentation by yeast S. cerevisiae) had the maximum response values. However, the response values of the samples fermented for 0–48 h (only fermented by lactic acid bacteria) were small. It means that lactic acid bacteria fermentation has little effect on the smell of the fermented beverages. What can be observed is that responding values of sensors increased remarkably for 60 h fermentation samples. Furthermore, the response values of S1, S5, S10, S13 sensors were all exceeded 2. Specific description of the gas sensor array in e-nose was mainly referred to the work reported by Chen, Zhang, Bhandari, and Guo (2018). It is said that S1, S5 and S13 sensors are sensitive to aromatic compounds (a class of gas components with
3.2. Changes in pH, titratable acidity, soluble solids, number of viable cells of lactic acid bacteria and yeast S. cerevisiae The pH, titratable acidity, soluble solids, number of viable cells of lactic acid bacteria and yeast S. cerevisiae of the unfermented substrate and fermented beverages can reflect the change of micro-ecological environment during fermentation. As shown in Table 2, before the fermentation, there was no significant difference (P > 0.05) in pH, titratable acidity, soluble solids among diverse samples. The initial
Table 2 pH, titratable acidity, oBrix, lactic acid bacteria and yeast cell count of unfermented substrate and fermented C. militaris beverage with different enzymatic hydrolysis. Treatments
Unfermented substrate
Fermented C. militaris beverage (after fermentation)
Control Cellulase/pectinase Cellulase/pectinase Cellulase/pectinase Control Cellulase/pectinase Cellulase/pectinase Cellulase/pectinase
pH
= 3:2 = 1:1 = 2:3 = 3:2 = 1:1 = 2:3
4.71 4.72 4.71 4.72 3.60 3.53 3.48 3.44
± ± ± ± ± ± ± ±
0.02a 0.01a 0.01a 0.01a 0.01A 0.01B 0.02C 0.01D
Titratable acidity (g/L)
0
2.82 ± 0.09a 2.94 ± 0.21a 3.13 ± 0.13a 3.01 ± 0.04a 10.60 ± 0.19A 10.66 ± 0.23A 10.74 ± 0.13A 10.82 ± 0.15A
16.65 16.85 17.15 17.05 15.55 15.85 15.65 15.70
Brix
± ± ± ± ± ± ± ±
0.05b 0.05ab 0.05a 0.15a 0.05A 0.05A 0.25A 0.10A
Lactic acid bacteria cell count (107 CFU/mL)
Yeast cell count (107 CFU/mL)
8.01 ± 0.12a 8.00 ± 0.09a 8.03 ± 0.07a 8.09 ± 0.11a 769.66 ± 24.32B 860.88 ± 60.15AB 926.11 ± 23.79A 934.15 ± 21.66A
N.D. N.D. N.D. N.D. 8.43 8.52 8.57 8.63
N.D., not detected. Different letters (a, b or A, B) in the same column indicate significant difference (p < 0.05). 4
± ± ± ±
0.60A 0.40A 0.50A 0.52A
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Fig. 3. Radar chart (a) and principal component analysis plot (b) about the changes of aroma of fermented C. militaris beverages obtained with different enzymatic hydrolysis (■: Control; ●: Cellulase/Pectinase 3:2; ▲: Cellulase/ Pectinase 1:1; ▼: Cellulase/Pectinase 2:3).
Fig. 2. Radar chart (a) and principal component analysis plot (b) about the changes of aroma of C. militaris fermentation broth during the whole fermentation process (■: 0 h; ●: 12 h; ▲: 24 h; ▼: 36 h; ♦: 48 h; ★: 48 + 12 h).
smaller odor difference among the enzymatic hydrolysis samples. Fermentation microorganisms of enzymatic hydrolysis group were more active than that of the control group (Table 2), which might produce more and richer volatile substances. Besides that, Urbonaviciene et al. pointed out the fermenting microorganisms can enhance the aroma of the fermented beverage (Urbonaviciene, Viskelis, Bartkiene, Juodeikiene, & Vidmantiene, 2015).
the pleasant scent). Moreover, S5 is sensitive to esters with the aromatic odor. It can be speculated that after two-step fermentation, esterification took place in the fermentation broth, which made the volatile flavor of the fermentation broth more prominent. And this is agreeable with Chan and Liu's (2019) research results. Fig. 2 (b) presents the principal component analysis (PCA) among the samples fermented at different time. There was little difference among the samples fermented for 0–48 h on the aroma, while the difference between the samples fermented for 60 h and that for 0–48 h was visible, which verified that the fermented C. militaris beverages would have more prominent volatile flavor after the subsequent yeast fermentation. All in all, two-step fermentation can make the beverage obtain a richer odor than before. Fig. 3 shows the effect of different enzymatic hydrolysis pretreatments on the volatile flavor components of fermented C. militaris beverages. As can be seen in Fig. 3 (a), compared with the control group, the enzymatic hydrolysis group had significantly higher response values on a series of sensors expect the S3, S4 and S14 sensors. The S1 response values with different ratios of enzymatic hydrolysis were all over 3 and the response value of S5 sensor with cellulase/pectinase of 1:1 was also above 3, which indicated that the enzymatic hydrolysis pretreatment could make the fermented beverage to have a rich aroma. The PCA presented in Fig. 3(b) more clearly shows that there was a significant difference (P < 0.05) in aroma between the control group and the enzymatic hydrolysis group, and the discrimination index (DI) of PCA was 90.8%. Moreover, the location of each enzymatic hydrolysis sample was much closer when compared with the control, which implied
3.3.2. E-tongue analysis E-tongue can convert electrical signals into taste signals to distinguish the taste of the fermented beverages, which has a small threshold of sensation and can well exclude the subjectivity of sensory evaluation (Jiang, Zhang, Bhandari, & Adhikari, 2018). Fig. 4 (a) displays the taste changes of C. militaris fermentation broth without enzymatic hydrolysis for 0 h, 12 h, 24 h, 36 h, 48 h and 60 h. It can be seen from the figure that with the fermentation time prolonging, the sourness of the fermentation broth gradually increased, the bitterness gradually decreased, while the signal values of the remaining taste sensors did not change obviously. The reduction of bitterness may be due to the formation of large amounts of acids masking other tastes. Furthermore, yeast fermentation produced small amount of alcohol, providing a mellow sense of the beverage (Lorenzini, Simonato, Slaghenaufi, Ugliano, & Zapparoli, 2019). Fig. 4 (b) represents the effect of different enzymatic hydrolysis pretreatments on the taste of the C. militaris beverages after two-step fermentation. As is shown in the picture that the enzymatic hydrolysis treatments had some effects on the sourness, 5
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Fig. 5. The principal component analysis (PCA) plot about the changes of taste of fermented C. militaris beverages obtained with different enzymatic hydrolysis ( : Control; ■: Cellulase/Pectinase 3:2; ▲: Cellulase/Pectinase 1:1; ★: Cellulase/Pectinase2:3).
3.3.3. Sensory analysis As can be seen in Table 3, the total score of the enzymatic hydrolysis group was significantly higher than that of control group, and all the scores of enzymatic hydrolysis group exceeded 80 points. The sensory scores for appearance were similar among the samples. The aroma scores were higher in the enzymatic group than that in the control group, and the highest scores were obtained in the cellulase/pectinase of 1:1. In the previous e-nose analysis, the cellulase/pectinase of 1:1 had the moderate aroma response values (Fig. 3 (a)). Sweetness and sourness may be related to the 0Bix, pH value and soluble solids. Concerning the consistency of beverages, the difference is not significant, which means the beverages are all uniform and stable. All in all, compared to the control group, the flavor of the enzymatic group was improved (see Table 4).
3.4. Nutritional quality analysis Fig. 4. Radar charts performed with e-tongue date. (a) Fermentation broth with different fermentation time (■: 0 h; ●: 12 h; ▲: 24 h; ▼: 36 h; ♦: 48 h; ★: 48 + 12 h); (b) Fermented beverages completed two-step fermentation with different enzymatic hydrolysis treatments (■: Control; ●: Cellulase/Pectinase 3:2; ▲: Cellulase/Pectinase 1:1; ▼: Cellulase/Pectinase 2:3).
3.4.1. Cordycepin The content of cordycepin in the fermented C. militaris beverage can reflect its nutritional quality (Jin et al., 2018). We could conclude from Fig. 6 (a) that the content of cordycepin increased significantly (P < 0.05) after enzymatic hydrolysis treatments, while there was no significant difference (P > 0.05) among different ratios of enzymes. Although the fermentation process would reduce the cordycepin, the group of cellulase/pectinase with the ratio of 2:3 had excellent retention (as high as 98.02%) of cordycepin. Regarding the group of cellulase/pectinase with the ratio of 3:2, it had the high content of cordycepin in the unfermented substrate with low retention of cordycepin after fermentation, but the content was still higher than that of the control group. From the above analysis, the enzymatic group with the cellulase/pectinase of 1:1 and 2:3, both on the quantity and retention of cordycepin were significantly better (P < 0.05) than that of the control group. Overall, enzymatic treatments had a good effect on cordycepin retention of C. militaris fermented beverage. According to the above analysis, there were more nutrients available to the microorganisms in the fermentation broth after enzymatic hydrolysis, which may protect the cordycepin from decomposition.
bitterness and astringency of the fermented beverages. The maximum difference in response values of sourness, bitterness and astringency between the control and enzymatic group was 0.4, 0.14 and 0.84, respectively. The most dominant difference was the astringency value of 3:2 group bigger than other samples. Fig. 5 shows the principal component analysis of fermented beverages between the control group and enzymatic hydrolysis group. According to Fig. 5, samples with cellulase/pectinase of 1:1 and 2:3 were close to the control group. In addition, the sample with cellulase/ pectinase of 3:2 separated from the control group obviously, which implicated the taste of these two samples were the most different. In general, the data points are scattered and interactive. It represents that the enzymatic hydrolysis has little effect on the taste of the fermented drinks.
6
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Table 3 Sensory analysis results of samples prepared by different enzymatic hydrolysis treatments. Treatments Control Cellulase/pectinase = 3:2 Cellulase/pectinase = 1:1 Cellulase/pectinase = 2:3
Appearance 16.14 16.41 16.91 15.91
± ± ± ±
Aroma b
0.58 0.39b 0.30a 0.33b
14.51 17.41 18.11 16.81
Sweetness ± ± ± ±
c
1.15 0.42ab 0.42a 0.70b
15.20 16.10 15.25 17.51
± ± ± ±
Sourness c
0.80 0.37b 0.85c 0.64a
16.11 15.30 16.21 15.94
± ± ± ±
Consistency a
0.74 0.32b 0.56a 0.49ab
14.90 15.51 15.61 16.37
± ± ± ±
Total scores b
1.30 0.62ab 0.66ab 1.60a
76.87 80.74 82.11 82.56
± ± ± ±
1.75c 1.22b 1.07ab 0.89a
Different letters (a, b, c) of the same column indicate significant differences (P < 0.05). Table 4 Retention of cordycepin in the fermented C. militaris beverages with different enzymatic hydrolysis treatments. Cellulase/Pectinase
Control
3:2
1:1
2:3
Retention of cordycepin (%)
88.84 ± 5.11c
79.94 ± 2.96d
90.38 ± 2.89b
98.02 ± 0.36a
One-way analysis of variance (n = 3) at 95% confidence level with different superscript letters (a, b, c, d) indicating a significant difference.
Fig. 6. Nutritional quality of the fermented C. militaris beverages: (a) Changes of cordycepin in unfermented and fermented C. militaris beverages with different : Unfermented substrate; : Fermented beverages), (b) hydroxyl radical scavenging rate % ( ) and (c) reducing power enzymatic hydrolysis treatments ( ) of the two-step fermented beverages with different enzymatic hydrolysis treatments. (A700nm: 7
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
3.4.2. Antioxidant activity Antioxidant activity is also an important quality parameter of the fermented beverage. The antioxidant capacity of fermented C. militaris beverage was evaluated by the hydroxyl radical scavenging rate (%) and reducing power (A700nm) of the fermentation broth. From the results presented in Fig. 6 (b) and Fig. 6 (c), it can be found that the enzymatic hydrolysis pretreatment can significantly (P < 0.05) improve the antioxidant capacity of fermented C. militaris beverage. When compared with the control, enzymatic hydrolysis group with the cellulase/pectinase of 3:2, 1:1 and 2:3 increased the hydroxyl radical scavenging rate by 14.20%, 16.08% and 28.76%, and improved the reducing power by 3.67%, 2.06% and 6.91%, respectively. This is presumably because enzymatic hydrolysis treatments increased the content of reducing sugars and cordycepin in the fermentation broth, which made the fermentation broth achieve more powerful antioxidant capacity. The antioxidant analysis is similar to the previously reported by Wu, Zhang, and Bhandari (2019).
Blandino, A., Al-Aseeri, M. E., Pandiella, S. S., Cantero, D., & Webb, C. (2003). Cerealbased fermented foods and beverages. Food Research International, 36(6), 527–543. Cai, L., Xiong, X., Lu, Z., & Sun, X. (2012). Purification and mutagenesis of Glucoamylase production strain and making technology of composite Koji. Food and Fermentation Technology, 48(6), 20–23. Champagne, C. P., Gomes da, C. A., & Daga, M. (2018). Strategies to improve the functionality of probiotics in supplements and foods. Current Opinion in Food Science, 22, 160–166. Chan, W., & Liu, S. (2019). The effects of carbohydrase, probiotic Lactobacillus paracasei and yeast Lindnera saturnus on the composition of a novel okara (soybean residue) functional beverage. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 100, 196–204. Chen, H. Z., Zhang, M., Bhandari, B., & Guo, Z. (2018). Evaluation of the freshness of fresh-cut green bell pepper (Capsicum annuum var. grossum) using electronic nose. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 87, 77–84. Chong, S. Y., & Wong, C. W. (2015). Production of spray-dried Sapodilla (Manilkara zapota) powder from enzyme-aided liquefied puree. Journal of Food Processing and Preservation, 39(6), 2604–2611. Das, S. K., Masuda, M., Sakurai, A., & Sakakibara, M. (2010). Medicinal uses of the mushroom Cordyceps militaris: Current state and prospects. Fitoterapia, 81(8), 961–968. Feng, Y., Zhang, M., Mujumdar, A. S., & Gao, Z. (2017). Recent research process of fermented plant extract: A review. Trends in Food Science & Technology, 65, 40–48. Granato, D., Putnik, P., Kovačević, D. B., et al. (2018). Trends in chemometrics: Food authentication, microbiology, and effects of processing. Comprehensive Reviews in Food Science and Food Safety, 17(3), 663–677. Guo, J., Yan, Y., Wang, M., Wu, Y., Liu, S. Q., Chen, D., et al. (2018). Effects of enzymatic hydrolysis on the chemical constituents in jujube alcoholic beverage fermented with Torulaspora delbrueckii. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 97(May), 617–623. Hancioǧlu, Ö., & Karapinar, M. (1997). Microflora of Boza, a traditional fermented Turkish beverage. International Journal of Food Microbiology, 35(3), 271–274. Jiang, H., Zhang, M., Bhandari, B., & Adhikari, B. (2018). Application of electronic tongue for fresh foods quality evaluation: A review. Food Reviews International, 34(8), 746–769. Jin, Y., Meng, X., Qiu, Z., Su, Y., Yu, P., & Qu, P. (2018). Anti-tumor and anti-metastatic roles of cordycepin, one bioactive compound of Cordyceps militaris. Saudi Journal of Biological Sciences, 25(5), 991–995. Lim, J. Y., Yoon, H. S., Kim, K. Y., Kim, K. S., Noh, J. G., & Song, I. G. (2010). Optimum conditions for the enzymatic hydrolysis of citron waste juice using response surface methodology (RSM). Food Science and Biotechnology, 19(5), 1135–1142. Lorenzini, M., Simonato, B., Slaghenaufi, D., Ugliano, M., & Zapparoli, G. (2019). Assessment of yeasts for apple juice fermentation and production of cider volatile compounds. Lwt- Food Science and Technology, 99, 224–230. Marsh, A. J., Hill, C., Ross, R. P., & Cotter, P. D. (2014). Fermented beverages with healthpromoting potential: Past and future perspectives. Trends in Food Science & Technology, 38, 113–124. Masuda, M., Urabe, E., Sakural, A., & Sakakibara, M. (2006). Production of cordycepin by surface culture using the medicinal mushroom Cordyceps militaris. Enzyme and Microbial Technology, 39, 641–646. Ma, L., Zhang, M., Bhandari, B., & Gao, Z. (2017). Recent developments in novel shelf life extension technologies of fresh-cut fruits and vegetables. Trends in Food Science and Technology, 64, 23–38. Oyaizu, M. (1986). Antioxidant activity of browning products of glucosamine fractionated by organic solvent and thin-layer chromatography. Nippon Shokulin Kogyo Gakkaishi, 35, 771–775. Palmqvist, E., & Hahn-Hägerdal, B. (2000). Fermentation of lignocellulosic hydrolysates. II: Inhibitors and mechanisms of inhibition. Bioresource Technology, 74(1), 25–33. Phan, C. W., David, P., Tan, Y. S., Naidu, M., Wong, K. H., Kuppusamy, U. R., et al. (2014). Intrastrain comparison of the chemical composition and antioxidant activity of an edible mushroom, pleurotus giganteus, and its potent neuritogenic properties. Scientific World Journal, 1–10 2014. Phan, C. W., Wang, J. K., Cheah, S. C., Naidu, M., David, P., & Sabaratnam, V. (2018). A review on the nucleic acid constituents in mushrooms: Nucleobases, nucleosides and nucleotides. Critical Reviews in Biotechnology, 38(5), 762–777. Sharma, H. P., Patel, H., & Sugandha (2017). Enzymatic added extraction and clarification of fruit juices: A review. Critical Reviews in Food Science and Nutrition, 57(6), 1215–1227. Smirnoff, N., & Cumbes, Q. J. (1989). Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry, 28(4), 1057–1060. Urbonaviciene, D., Viskelis, P., Bartkiene, E., Juodeikiene, G., & Vidmantiene, D. (2015). The use of lactic acid bacteria in the fermentation of fruits and vegetables - technological and functional properties. Biotechnology, 135–164 April. Wang, Z., Wang, R., Wu, Y., Zhou, F., Hua, C., Gu, Z., et al. (2014). Optimization of the extraction Cordyceps militaris polysaccharides by microwave assisted with enzyme hydrolysis. Science and Technology of Food Industry, 35(11), 188–192. Woraharn, S., Lailerd, N., Sivamaruthi, B. S., Wangcharoen, W., Sirisattha, S., Peerajan, S., et al. (2016). Evaluation of factors that influence the L-glutamic and γ-aminobutyric acid production during Hericium erinaceus fermentation by lactic acid bacteria. CyTA - Journal of Food, 14(1), 47–54. Wu, X., Zhang, M., & Bhandari, B. (2019). A novel Infrared Freeze drying ( IRFD ) technology to lower the energy consumption and keep the quality of cordyceps militaris. Innovative Food Science & Emerging Technologies, 54, 34–42. Yang, R., Gu, D., & Gu, Z. (2016). Cordyceps rice wine: A novel brewing process. Journal of Food Process Engineering, 39(6), 581–590. Zhang, M., Chen, H., Mujumdar, A. S., Tang, J., Miao, S., & Wang, Y. (2017). Recent
4. Conclusions A nutrient-rich fermented C. militaris beverage that is dark reddishbrown, clear and transparent, sweet and sour was obtained. Enzymatic hydrolysis can produce more reducing sugars, which is conducive to the subsequent fermentation process. Two-step fermentation with lactic acid bacteria and yeasts yielded a pleasant fragrant and aromatic odor, making the fermented beverage acquire a pleasant taste. Besides, enzymatic hydrolysis treatments could enhance the volatile flavor profile of fermented beverages. With the use of cellulase/pectinase ratio of 2:3, cordycepin was extracted more in the unfermented substrate and had a better retention after fermentation. The enzymatic treatments also significantly improved the antioxidant activity of the fermented C. militaris beverage. Overall, enzymatic hydrolysis pretreatment with fermentation provided a promising method for improving the flavor and enhancing the nutritional quality of fermented beverage. The shelflife of this functional fermented beverage will be the subject of future study. Author contributions Literature research and experiments were performed by Yanyan Lao. Oversight of the project and field experience were provided by Min Zhang, Zhongqin Li, and Bhesh Bhandari. Critical review of the manuscript was performed by Min Zhang and Bhesh Bhandari. Declaration of competing interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript. Acknowledgments We acknowledge the financial support from the National Key R&D Program of China (Contract No. 2018YFD0400801), the 111 Project (BP0719028), the Yangzhou City Agricultural Key R&D Program (No.YZ2019034), Jiangsu Province (China) “Collaborative Innovation Center for Food Safety and Quality Control” Industry Development Program and the National First-class Discipline Program of Food Science and Technology (No. JUFSTR20180205), all of which enabled us to carry out this study. References Altay, F., Karbancioglu-Güler, F., Daskaya-Dikmen, C., & Heperkan, D. (2013). A review on traditional Turkish fermented non-alcoholic beverages: Microbiota, fermentation process and quality characteristics. International Journal of Food Microbiology, 167(1), 44–56.
8
LWT - Food Science and Technology 120 (2020) 108934
Y. Lao, et al.
Zhang, M., Li, C. L., & Ding, X. L. (2005). Effects of heating conditions on the thermal denaturation of white mushroom suitable for dehydration. Drying Technology, 23(5), 1119–1125. Zhang, M., Zhang, C. J., & Shrestha, S. (2005). Study on the preparation technology of superfine ground powder of Agrocybe chaxingu Huang. Journal of Food Engineering, 67(3), 333–337.
developments in high-quality drying of vegetables, fruits and aquatic products. Critical Reviews in Food Science and Nutrition, 57(6), 1239–1255. Zhang, Y., Fraatz, M. A., Birk, F., Rigling, M., Hammer, A., & Zorn, H. (2018). Enantiomeric ratios of 2-methylbutanoic acid and its methyl ester: Elucidation of novel biogenetic pathways towards (R)-methyl 2-methylbutanoate in a beverage fermented with shiitake. Food Chemistry, 266(June), 475–482.
9