Regulating yeast flavor metabolism by controlling saccharification reaction rate in simultaneous saccharification and fermentation of Chinese Maotai-flavor liquor

International Journal of Food Microbiology 200 (2015) 39–46

Contents lists available at ScienceDirect

International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Regulating yeast flavor metabolism by controlling saccharification reaction rate in simultaneous saccharification and fermentation of Chinese Maotai-flavor liquor Qun Wu 1, Bi Chen 1, Yan Xu ⁎ State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China

a r t i c l e

i n f o

Article history: Received 19 September 2014 Received in revised form 3 January 2015 Accepted 15 January 2015 Available online 22 January 2015 Keywords: Chinese liquor Filamentous fungi Mixed-culture fermentation Simultaneous saccharification and fermentation Yeast

a b s t r a c t Maotai-flavor liquor is produced by simultaneous saccharification and fermentation (SSF), in which filamentous fungi produce hydrolases to degrade the starch into fermentable sugar. Saccharomyces cerevisiae simultaneously transforms the sugars to ethanol and flavor compounds. The saccharification rate plays an important role in regulating the liquor yield and flavor profile. This work investigated the effect of saccharification rate on fermentation by regulating the inoculation ratio (1:0.1, 1:0.5, 1:1, 1:5, 1:10) of S. cerevisiae and Aspergillus oryzae, the main saccharification agent. We found no significant difference in reducing sugar content among the mixed cultures with different ratios. This indicated a balance of the saccharification rate and the sugar consumption rate, in which the former was controlled by the interaction between A. oryzae and S. cerevisiae, and the latter controlled the metabolism of the two species. The ethanol yield was the highest in ratios of 1:0.5, 1:1, and 1:5, while the total production of flavor compounds was the highest for the ratio of 1:0.5, which was mainly attributed to the vigorous metabolism of S. cerevisiae. The inoculum ratio of 1:10 produced the second highest content of flavor compounds in which a large number of alcohols and esters were derived from the vigorous metabolism of A. oryzae. This indicated that the saccharification rate significantly influenced the flavor metabolism. This study improves understanding of the interaction and cooperation between A. oryzae and S. cerevisiae in co-culture fermentation for Chinese liquor making. © 2015 Published by Elsevier B.V.

1. Introduction Simultaneous saccharification and fermentation (SSF) is commonly used to produce many types of traditional fermented food and beverages in Asian countries (Chen et al., 2014; Furukawa et al., 2013; Li et al., 2013). The method combines the saccharification of starch to fermentable sugar and the sugar to different compounds, including ethanol and other compounds (Olofsson et al., 2008). The approach of SSF is different from the processes used to prepare some other alcohol beverages such as beer, which is made by separate hydrolysis and fermentation (SHF) processes (Farías et al., 2010; van Beek and Priest, 2002). During SSF, starch should be hydrolyzed to fermentable sugar by hydrolytic enzymes, such as α-amylase and glucoamylase, which could be produced by many fungal species, such as Aspergillus oryzae,

Abbreviations: SSF, simultaneous saccharification and fermentation; SHF, separate hydrolysis and fermentation; FAN, free amino nitrogen. ⁎ Corresponding author. Tel./fax: +86 510 85864112. E-mail address: [email protected] (Y. Xu). 1 The two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.01.012 0168-1605/© 2015 Published by Elsevier B.V.

Paecilomyces varioti, Rhizopus sp., Monascus sp., and Penicillium sp. Therefore, these filamentous fungi have been reported to serve as the saccharifying agents (Chen et al., 2014; Nahar et al., 2008; Lv et al., 2012), while Saccharomyces cerevisiae has served as a fermenting agent to convert fermentable substrates into ethanol. The main benefit of the SSF process, when compared with a SHF process, is less inhibition by reducing sugars in the enzymatic hydrolysis, which increases the efficiency of substrate utilization and improves the ethanol yield (Olofsson et al., 2008). Therefore, SSF methods have attracted considerable attention all over the world, although these processes have some drawbacks. It has been generally accepted that the main drawback of SSF is the different conditions (temperature and pH) that are required for optimization of the two reactions (Lee et al., 2012; Lin and Tanaka, 2006). However, the most important factor is the regulation of the coordination of the two reactions. This is because different saccharification rates will produce different sugar concentrations, thereby significantly influencing cell growth and the metabolism of ethanol and flavor compounds. This ultimately affects the final ethanol yield and quality of the food. Flavor profile is the most important characteristic of fermented food, and it has been regulated by many methods. For example, flavor profile

40

Q. Wu et al. / International Journal of Food Microbiology 200 (2015) 39–46

can be influenced by the nitrogen concentration of the substrate (Lage et al., 2014), while other studies have added several non-Saccharomyces to regulate the flavor profile (Medina et al., 2013). However, the effect of saccharification rate on the flavor metabolism of yeast has never been investigated, even though this process is important for flavor production in SSF processes. The microbial interaction between fungi and yeast is important for regulation of the coordination of these two reactions. These microorganisms are considered to interact and cooperate with each other in various natural environments (Murado et al., 2008). There are a few studies concerning the interaction between filamentous fungi and yeasts in co-culture fermentation. Shin et al. (1998) reported that the biomass and pigment production of Monascus spp. increased significantly when co-cultured with S. cerevisiae. More recently, Zhou et al. (2011) showed that the polygalacturonase production of Aspergillus niger improved significantly when mixed with S. cerevisiae J-1. Ge et al. (2013) found that enzyme production of S. cerevisiae and the ethanol conversion rate increased significantly in co-culture with A. oryzae. In addition, it was found that A. oryzae was beneficial for flavor production in SSF of rice wine (Yang et al., 2013). These studies indicated that filamentous fungi and yeast have a positive interaction in co-culture fermentation. Maotai-flavor liquor is a symbolic drink in China just as whisky in Scotland, and brandy in France, and is produced by a complicated spontaneous fermentation process (Wu et al., 2013). It includes Daqu (the starter) making, stacking fermentation and liquor fermentation stages. After Daqu making stages, the ground Daqu and steamed sorghum are mixed and piled up on the ground for stacking fermentation. When the temperature on the top of the stack reaches about 50 °C, the mixture is then put into the underground cubic pits, and sealed for liquor fermentation for about 30 days (Wu et al., 2013). It is also a typical SSF process, and accumulates a specific microorganism community, including filamentous fungi and yeasts. In stacking fermentation, the population of filamentous fungi and yeast increased from 3.4 × 103 to 5.8 × 103 CFU/g (Chen et al., 2014), and from 0.37 to 7.97 × 105 CFU/g at the top of the stack (Wu et al., 2013), respectively. While in the upper layer of liquor fermentation, the population of filamentous fungi continuously decreased and few fungal species survived after 10 days (Chen et al., 2014), while the yeast kept stable and quickly decreased after 10 days (Wu et al., 2013). The co-existence of filamentous fungi and yeast indicated that they could interact in the whole stage of stacking fermentation and the early stage of liquor fermentation, which might be important for the SSF process of Maotai-flavor liquor making. In the whole liquor making process, eight and seven different fungal species were identified by using culture-dependent and cultureindependent methods of analysis, respectively. Among them, A. oryzae was the predominant species; it exhibited high α-amylase and glucoamylase activities, indicating that it plays an important role in producing amylase for hydrolyzing the starch (Chen et al., 2014). A. oryzae is an important type of filamentous fungus that is widely used in east Asian foods and liquor fermentation (Furukawa et al., 2013). This study investigated the interactions and cooperation between A. oryzae and S. cerevisiae, and the effect of the saccharification rate on ethanol and flavor metabolism. This work examines the metabolism processes of SSF, and furthers understanding of the fermentative mechanism for preparation of traditional fermented food. 2. Materials and methods 2.1. Microorganisms and medium A. oryzae and S. cerevisiae were isolated from the Maotai-flavor liquor making process at a facility in Guizhou Province, by the method of Rose Bengal Agar medium and WLN medium as described in Chen et al. (2014), and Wu et al. (2013), respectively. They were deposited in the

China General Microbiological Culture Collection Center with accession numbers CGMCC 6264 and CGMCC 4747, respectively. 2.2. Mixed-culture fermentations For yeast starter culture, a loopful of yeast culture was inoculated into a 250-ml Erlenmeyer flask containing 50 ml of liquid sorghum extract, which is used as the main starting material for Chinese liquor production. Two hundred grams of sorghum powder was added with 800 ml of deionized water, steamed for 2 h and then saccharified at 60 °C for 4 h with the addition of glucoamylase at a final concentration of 50 U/g. The supernatant was selected as the final sorghum extract by centrifugation at 8000 g for 15 min. The final reducing sugar content of the liquid sorghum extract was about 90 mg/g, and was mainly glucose. No nitrogen was added, and the initial pH was kept unadjusted, which was about 6.0. Fermentations were conducted at 30 °C for 48 h with stirring at 150 rpm. For filamentous fungi starter culture, the spores of A. oryzae were harvested after 3–5 days of culture on potato dextrose agar (PDA) medium at 28 °C. The PDA medium was prepared from 200 g washed and sliced potatoes, boiled in 500 ml deionized water and strained through gauze, then 20 g glucose, 20 g agar and another 500 ml deionized water were added. After the preparation of these two starters, their cell numbers were calculated using a hemocytometer, and the inoculum concentrations were adjusted for the mixed culture. The population of yeast starter was about 5 × 108 CFU/ml, while the initial concentration of fungi spore suspension was about 5 × 109 CFU/ml, and the suspension was gradient diluted and added to the solid-fermentation medium, to keep the same volume of the added liquid. The mixed culture was carried out in the solid-state fermentation medium, which was also prepared with sorghum. Sorghum was ground and soaked in hot water for 12 h. Then 200 g of sorghum containing 50% water was sealed in a flask and autoclaved for 30 min at 121 °C. To achieve limited aerobic conditions, flasks were fitted with a one-hole silicon stopper into which a cotton-plugged Pasteur pipette was inserted to vent CO2 during fermentation. To determine the effect of environmental factors on co-culture fermentation, A. oryzae (5 × 105 spores/g) and S. cerevisiae (5 × 105 CFU/g) were inoculated and fermented at different temperatures (25, 30, 35, and 40 °C) at pH 5.0, and at different pH (3.5, 4.0, 4.5, 5.0) at 30 °C. For the effect of reducing sugar content on single culture fermentation of S. cerevisiae, the initial sugar content in sorghum solid-state fermentation medium was controlled at 50 mg/g, and glucose was added to adjust the sugar content to different concentrations (50, 100, 150, and 200 mg/g). For the effect of microbial interaction of the two species, S. cerevisiae (5 × 105 cells/g) was mixed with A. oryzae spores at ratios of 1:0.1, 1:0.5, 1:1, 1:5, 1:10, at 30 °C and pH 4.5. The fermentations were conducted for 15 days and sampling was carried out at 0, 2, 4, 6, 9, 12, and 15 days. The samples were stored at 4 °C for cell counting and − 20 °C for chemical analysis. A noninoculated sample of fermentation medium was prepared as the control. All experiments were performed in triplicate. 2.3. Chemical and biomass analysis of samples in liquor fermentation Samples (10 g) were mixed with 90 ml of distilled water, ultrasonicated at 0 °C for 30 min, and centrifuged at 4 °C for 5 min. The obtained supernatant was used to determine the content of reducing sugars and ethanol. Reducing sugar was measured by the method of DNS (3,5-dinitrosalicylic acid) as described in Wang et al (2013). It was mainly glucose in this sorghum extract. The ethanol content was determined by HPLC (Agilent) using a column Aminex HPX-87H (Bio-Rad). The column was eluted at 60 °C with a degassed mobile phase containing 3 mM H2SO4 at a flow rate of 0.6 ml/min.

Analysis of variance was conducted using an ANOVA Tukey's test to determine any significant difference between samples. The statistical level of significance was set at P b 0.05. Principal component analysis (PCA) was also carried out on the concentration of volatile compounds in order to visualize relationships between variables and between samples and variables. 3. Results and discussion 3.1. Influence of initial reducing sugar on single culture fermentation of S. cerevisiae To examine the effect of reducing sugar content on single culture fermentation of S. cerevisiae fermentation, sorghum solid-state fermentation medium was used. The initial sugar content was 50 mg/g, and was adjusted to different concentrations (50, 100, 150, and 200 mg/g) with different addition of glucose. Then S. cerevisiae was inoculated in these mediums, respectively. As shown in Fig. 1, although the highest ethanol concentration (79.5 mg/g) was obtained at the highest sugar concentration (200 mg/g), the yeast population was the lowest (3.63 × 106 CFU/g). In contrast, although the ethanol concentrations were only 21.5 and 43.3 mg/g at sugar levels of 50 and 100 mg/g, respectively, the yeast growth was the most efficient, with populations of 0.7 × 108 and 1.70 × 108 CFU/g. These results indicated that the sugar

80

8

60

6

40

4

20

2

Yeast biomass (lg CFU/g)

10

0

0 50

100

150

200

content significantly influenced the metabolism of the cells, and that higher concentrations were harmful to cell growth and not economic for fermentation. 3.2. Influence of environmental conditions on co-culture fermentation The environmental factors, such as temperature and pH, affect the performance of traditional liquor fermentation. Not only are these factors important indicators of microorganism growth and metabolism, they also affect the bioactivity of microorganism (Wu et al., 2013). Previous research has shown that the best temperature and pH for S. cerevisiae fermentation were about 30 °C and pH 4.0 (Lee et al., 2012), while the best temperatures for glucoamylase activity and

150

Residual glucose Ethanol content Yeast biomass

a 120

10 8

90

6

60

4

30

2

0

0 25

140 120

Yeast biomass (lg CFU/g)

2.5. Statistical analysis

Residual sugar Ethanol content Yeast biomass

b

30

35

Temperature (°C)

40

10

Residual glucose Ethanol content Yeast biomass

8

100 80

6

60

4

40 2

20

Yeast biomass (lg CFU/g)

Volatile compounds in the culture broth were assayed by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME–GC–MS). A 50/30 μm divinylbenzene/carboxen/ poly(dimethylsiloxane) (DVB/CAR/PDMS) coated fiber (Supelco, Bellefonte, PA, USA) was used for volatile compound extraction. HSSPME extraction and GC–MS analysis were performed according to the methods previously described (Wu et al., 2014). Triplicate analyses were performed for each sample.

100

Fig. 1. Effect of reducing sugar concentration on single culture fermentation of S. cerevisiae in solid-state sorghum medium.

Glucose and ethanol content (mg/g)

2.4. GC–MS analysis of volatile compounds

41

Initial sugar content (mg/g)

Glucose and ethanol content (mg/g)

Starch was extracted from samples by acid hydrolysis (20% HCl, v/v) for 30 min. After the pH value of the hydrolysate was adjusted to 7.0 with 20% (w/v) NaOH, the total reducing sugar was determined. Starch content was estimated by calculating the difference between total reducing sugar and the original reducing sugar. The free amino nitrogen (FAN) content in the solid-state fermentation sample was determined according to the ninhydrin method described by Morrall et al (1986), except 10 g of sample was used. The results were expressed as mg FAN/g dry weight. Yeast was enumerated by plate culture on WLN agar as described in Wu et al. (2013). Each sample (10 g) was mixed with 90 ml of sterile saline (0.85% NaCl) and soaked at 4 °C for 30 min, and then 100 μl of each dilution was spread on WLN medium in triplicate. Cultures were incubated at 30 °C for 4 days. All the samples were analyzed in triplicate. Real-time quantitative polymerase chain reaction (qPCR) was used to monitor the growth of A. oryzae in the fermentation. The primer AoFor/AoRev for A. oryzae, which also targeted the ITS region, was obtained from a previous study (Sardiñas et al., 2011). Each qPCR reaction was performed according to the description for SsoFast EvaGreen Supermix (Bio-Rad). The amplification conditions were: preheating at 98 °C for 2 min and then 40 cycles of 98 °C for 5 s, 57 °C for 5 s, and melting curve from 70 to 95 °C by increasing every 0.5 °C for 5 s (Bio-Rad CFX96, China). Glucoamylase activity was determined using the DNS method (Chen et al., 2014). Soluble starch (1%) was used as the substrate. An enzyme activity unit (U) was defined as the amount of enzyme that liberated 1 mg of reducing sugar per hour under assay conditions. All experiments were run in triplicate.

Glucose and ethanol content (mg/g)

Q. Wu et al. / International Journal of Food Microbiology 200 (2015) 39–46

0

0 3.5

4.0

pH

4.5

5.0

Fig. 2. Effect of fermentation conditions on co-culture of A. oryzae and S. cerevisiae. a, temperature; b, initial pH.

42

Q. Wu et al. / International Journal of Food Microbiology 200 (2015) 39–46

Chinese liquor fermentation stage, the temperature increased from about 25 °C to 38–39 °C in the pit (Wu et al., 2013), therefore, the two species evolved to survive and metabolize in a wide temperature spectrum, which would be important for the success of liquor making. The effect of pH on cell growth and ethanol production in co-culture fermentation is shown in Fig. 2b. Yeast biomass and ethanol concentration reached maximum values of 1.74 × 108 CFU/g and 124.7 mg/g, respectively, at pH 4.5, and the residual reducing sugar level decreased to the lowest level (19.3 mg/g). Cell growth and ethanol concentration remained high (2.63 × 107 CFU/g and 103.5 mg/g) at pH 4.0. As there is also an acidic condition during the liquor fermentation process (pH 4.0–5.0), these two species from this process are more likely to be active in acidic conditions. Therefore, they prefer slightly acidic conditions for growth and metabolism.

180

14

a

12

Glucoamylase activity (U/g)

Biomass of A. oryzae (×108 spores/g)

A. oryzae growth were about 60 °C and 28 °C, respectively (Slivinski et al., 2011). Because the two strains used in co-cultures do not always have similar culture requirements (Farid et al., 2002), it is very important to optimize the conditions for co-culture fermentation. The effect of temperature on the co-culture fermentation is shown in Fig. 2a. The yeast biomass and ethanol concentration reached maximum values of 1.17 × 108 CFU/g and 132.5 mg/g, respectively, at 30 °C. Yeast also consumed the most reducing sugar at 30 °C. These results indicate that the rate of saccharification of the starch and the rate of sugar fermentation are probably balanced at 30 °C, and that this temperature is optimal for yeast growth. Although yeast biomass and ethanol content decreased with increasing temperature, these values still reached 3.47 × 106 CFU/g and 63.7 mg/g at 40 °C. It indicated the high temperature-resistant activity of this S. cerevisiae and A. oryzae. In

1:0.1 1:0.5 1:1 1:5 1:10

10 8 6 4 2

b 150

1:0.1 1:0.5 1:1 1:5 1:10

120 90 60 30 0

0 0

2

4

6

8

10

12

14

0

16

2

1:0.1 1:0.5 1:1 1:5 1:10 control

100 90 80 70 60 50 40 30 20 10 0 0

2

Biomass of S.cerevisiae (×108 CFU/g)

Glucose concentration (mg/g)

c

210

4

6

8

10

6

8

10

12

12

14

5.0

14

16

1:0.1 1:0.5 1:1 1:5 1:10 control

d

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

16

2

4

6

8

10

12

14

16

Fermentation time (d)

Fermentation time (d)

Ethanol concentration (mg/g)

4

Fermentation time (d)

Fermentation time (d)

e

160

1:0.1 1:0.5 1:1 1:5 1:10 control

140 120 100 80 60 40 20 0 0

2

4

6

8

10

12

14

16

Fermentation time (d) Fig. 3. Effect of ratio of S. cerevisiae and A. oryzae on co-culture fermentation. a, A. oryzae growth; b, glucoamylase activity; c, reducing sugar content; d, S. cerevisiae growth; e, ethanol concentration.

Q. Wu et al. / International Journal of Food Microbiology 200 (2015) 39–46

3.3. Effect of ratio of S. cerevisiae and A. oryzae on co-culture fermentation The inoculum ratio is important for the interaction between fungi and yeast; thus, the effect of the ratio on co-culture fermentation was investigated. As shown in Fig. 3d and e, the population of S. cerevisiae and ethanol production were lowest in the single culture SHF process, reaching maximum values of 1.10 × 108 CFU/g and 80.1 mg/g at day 6. By contrast in SSF, the sugar concentration was kept at a suitable range to maintain gradual yeast growth and ethanol production for a long period, and consequently led to a high yield of ethanol. In the SSF process, the population of A. oryzae increased with increasing inoculation amount, and reached a maximum when its inoculum was tenfold that of S. cerevisiae. In addition, it reached its peak at day 2 and then gradually declined until the end of fermentation (Fig. 3a). The glucoamylase activity also increased with the population of A. oryzae and reached a maximum at day 2, which was consistent with the growth of A. oryzae. Glucoamylase activity reached a maximum value of 145 U/g at the ratio of 1:10 (Fig. 3b). The reducing sugar content was monitored during the fermentations, as shown in Fig. 3c. In single culture, the reducing sugar content decreased quickly from 207.7 to 104.5 mg/g in the first 2 days, and then decreased more slowly to a final concentration of 38.7 mg/g at the end of fermentation. The strong sugar consumption in the first 2 days indicated the vigorous growth of S. cerevisiae, which was a primary reason for its early death. In the different co-culture fermentations, the reducing sugar level increased in the first 2 days and reached a peak, ranging from 42.6 to 52.6 mg/g, after which the level gradually fell until the end of fermentation. This was a result of the low saccharifying rate caused by the inhibition of glucoamylase activity by the accumulation of ethanol and organic acids as fermentation progressed. Although the glucoamylase activity varied significantly between the SSF trials, the reducing sugar content showed no such difference, except that the reducing sugar level was slightly higher in the 1:10 trial. Because the reducing sugar content represents the balance between starch saccharification and sugar consumption, the sugar consumption rate is regulated by the saccharification rate. In turn, the saccharification rate is determined by the A. oryzae population and the glucoamylase activity. Therefore, it can be reasoned that the A. oryzae inoculum can determine the sugar releasing activity, and thereby regulate the growth and metabolism of S. cerevisiae. As shown in Fig. 3d, the growth dynamics of S. cerevisiae were different with the different inoculum ratios of A. oryzae. S. cerevisiae growth increased when the inoculation content of A. oryzae decreased, and reached maximum biomass at day 6 for inoculum ratios of 1:0.1, 1:0.5, and 1:1. Maximum biomass was reached earlier (day 4) for inoculum ratios of 1:5 and 1:10, and the maximum values were lower than those observed for the other ratios. Although the fungi provided fermentable sugar for S. cerevisiae, there also existed other kinds of interactions. When the inoculum amount of A. oryzae increased, the sugar content was nearly the same, which would not lead to inhibition of S. cerevisiae. Therefore, it appears that the inhibition of S. cerevisiae was a result of the biomass of A. oryzae. This indicates the competition of A. oryzae and S. cerevisiae, and shows that increasing the A. oryzae inoculum is not beneficial for S. cerevisiae growth. The consumption of reducing sugar by S. cerevisiae to produce ethanol is shown in Fig. 3e. A rapid exponential phase was observed initially and entered a stationary phase after about 6 days of fermentation in SSF. The highest ethanol concentration was obtained at the end of fermentation for the inoculum ratios of 1:0.5, 1:1, and 1:5, ranging from 140.46 to 145.94 mg/g. For the inoculum ratios of 1:0.1 and 1:10, the ethanol level reached 134.5 and 125.9 mg/g, respectively, at day 9, and then gradually decreased, indicating that the higher amounts of A. oryzae were not suitable for ethanol production. FAN was reported to have effect on yeast fermentation (Wu et al., 2010). Therefore, its concentration was also determined in these different cultures. The initial FAN concentration was 1.30 mg/g, and

43

the final concentrations were 1.22, 1.23, 1.33, 1.30 and 1.45 mg/g in cultures of 1:0.1, 1:0.5, 1:1, 1:5 and 1:10, respectively, at the end of fermentation. FAN concentration did not decrease after the fermentation, and there was no significant difference between these samples. This might be due to the strong activity of protease produced by A. oryzae. It indicated that FAN might not be an important factor for cell growth in these co-culture fermentations. The fermentation parameters in SSF and SHF fermentation are shown in Table 1. The specific growth rate and specific ethanol production rate were the lowest in single culture fermentation, indicating the efficiency of the SSF process. For SSF, all specific rates reached maximum values for the 1:0.1 inoculum ratio, including the specific growth rate of the two species, and the specific rates of ethanol production and enzyme activity. These rates all gradually decreased with increasing A. oryzae inoculum level. Because the biomass of A. oryzae was the lowest at the 1:0.1 ratio, it is expected that its inhibition on S. cerevisiae would be the lowest. Although the maximum specific rate of ethanol production was also obtained at the ratio of 1:0.1, the ethanol yield was not the highest. This is because the ethanol production period for this fermentation was shorter, possibly caused by an insufficient supply of sugar.

3.4. Effect of ratio of S. cerevisiae and A. oryzae on metabolic profiles in co-culture fermentation The effect of the inoculum ratio of S. cerevisiae and A. oryzae on metabolic production of aromatic compounds in co-culture fermentation was investigated. As shown in Table 2, a total of 29 aromatic compounds were quantified, including 14 esters, 9 alcohols, 2 acids, 2 aldehydes, and 2 ketones. A. oryzae produced only 2 esters (ethyl caproate and pentyl propionate) that would not be produced by S. cerevisiae in single and co-culture, indicating that it played little role in ester production in co-culture fermentation. In single fermentation, S. cerevisiae did not produce ethyl-2-methylbutyrate, 3-methylbutyl acetate, or ethyl nonanoate, and the concentration of each detected ester was significantly lower than those in co-culture fermentations. For example, the concentrations of ethyl acetate and ethyl 2-phenylacetate, which have pineapple, rose, and honey aromas (Fan and Qian, 2006), increased more than 50 times and 4 times, respectively, after co-culture fermentation with A. oryzae. This indicates that A. oryzae stimulates the metabolism of S. cerevisiae, and that the SSF process is beneficial for S. cerevisiae growth and metabolism. Cultures with inoculum ratios of 1:0.5 and 1:10 produced the highest levels of esters. Higher alcohols form another large class of volatiles found in single and co-culture fermentations. A. oryzae is an efficient higher alcohol producer; it produced five different alcohols, with a combined concentration of 8056.8 μg/kg in single culture. S. cerevisiae produced less higher alcohol than A. oryzae in single culture, and did not produce 1-hexanol, 3-octanol, or 1-octanol. However, it produced more alcohol in SSF than in SHF; for example, production of 3-methylbutanol and 2-phenethyl alcohol increased by more than tenfold and fivefold, respectively, in co-culture fermentations. The production of higher alcohols was highest with an inoculum ratio of 1:0.5. Table 1 Fermentation parameters in different SSF and SHF processes. Inoculation ratio (S. cerevisiae/A. oryzae)

1:0.1

1:0.5

1:1

1:5

1:10

Control

μ maxSC (/h) μ maxAO (/h) qpmax (mg/h/108 CFU) qEmax (U/h/108 spores)

12.90 70.10 479.75 774.51

9.30 15.52 459.32 250.63

10.05 8.92 478.51 88.83

10.83 1.67 462.13 19.20

8.65 0.78 417.11 14.51

1.45 – 183.98 –

μ maxSC: maximum specific growth rate of S. cerevisiae; μ maxAO: maximum specific growth rate of A. oryzae; qpmax: maximum specific production rate of ethanol; qEmax: maximum specific enzyme activity; Control, SHF process.

44

Table 2 Concentrations of flavor compounds produced in different SSF and SHF processes (μg/kg, n = 3). 1:0.1

1:0.5

1:1

1:5

1:10

S. cerevisiae

A. oryzae

Esters Ethyl acetate Ethyl 2-methylpropanoate Ethyl caproate Ethyl 2-methylbutyrate 3-Methylbutyl acetate Pentyl propionate Ethyl nonanoate Ethyl caproate Diethyl succinate Ethyl 2-phenylacetate Ethyl 3-phenylpropanoate Ethyl palmitate Ethyl oleate Ethyl linoleate ∑

2394.43 ± 329.15 1339.69 ± 237.34 – 85.27 ± 13.82 467.93 ± 56.66 – 16.27 ± 2.27 99.89 ± 16.29 6829.04 ± 684.42 3906.11 ± 389.65 347.48 ± 28.06 752.84 ± 32.95 165.99 ± 14.25 139.45 ± 20.27 16,544.39

3899.47 ± 650.00 2573.92 ± 353.90 – 165.59 ± 37.03 949.64 ± 104.32 – –a 38.68 ± 8.68 11,853.07 ± 844.25 8449.05 ± 730.98 793.43 ± 122.77 4595.39 ± 438.60 1129.74 ± 107.84 773.05 ± 113.97 35,221.03

2625.33 ± 129.59 2077.47 ± 163.44 – 109.53 ± 8.91 656.05 ± 56.81 – – 31.06 ± 3.06 7893.96 ± 666.82 5401.34 ± 157.83 492.95 ± 39.38 3795.90 ± 105.51 949.23 ± 66.14 509.87 ± 29.68 24,542.69

2373.86 ± 163.54 2696.76 ± 243.40 – 89.93 ± 4.48 452.34 ± 66.67 – – – 8857.11 ± 868.62 6361.04 ± 757.34 475.32 ± 27.00 4176.34 ± 817.03 1052.41 ± 197.88 493.47 ± 132.17 27,028.58

3443.39 ± 747.29 1744.34 ± 148.11 – 165.21 ± 29.11 555.31 ± 52.24 – 44.28 ± 4.28 46.91 ± 6.91 3629.48 ± 852.66 18,110.67 ± 2597.32 928.86 ± 134.29 5919.19 ± 792.85 1470.89 ± 187.31 750.88 ± 115.02 36,809.41

43.80 ± 8.34 273.94 ± 30.63 – – – – – 10.88 ± 1.53 210.75 ± 19.43 957.11 ± 84.74 68.91 ± 8.36 907.53 ± 81.17 224.81 ± 34.54 126.43 ± 23.54 2824.16

– – 116.45 ± 9.44 – – 113.47 ± 6.37 – – – – – – – – 229.92

Alcohols 2-Methylpropanol 3-Methylbutanol 1-Hexanol 3-Octanol 1-Octen-3-ol 1-Octanol 2,3-Butanediol trans-2-Undecen-1-ol 2-Phenethyl alcohol ∑

647.32 ± 86.34 4107.07 ± 364.20 – 84.36 ± 14.60 4150.18 ± 365.77 476.58 ± 49.94 301.49 ± 27.85 141.07 ± 15.01 9122.45 ± 485.81 19,030.52

939.60 ± 160.28 7076.37 ± 755.87 – 159.10 ± 84.49 7061.45 ± 356.34 790.30 ± 104.19 577.63 ± 62.38 229.78 ± 40.64 18,864.83 ± 233.27 35,699.06

639.89 ± 25.54 4517.22 ± 359.24 – 104.21 ± 7.04 4763.91 ± 69.52 589.60 ± 25.04 541.83 ± 36.12 165.09 ± 40.49 12,100.05 ± 835.84 23,421.8

757.34 ± 55.89 4819.47 ± 235.01 – 44.86 ± 4.86 4661.94 ± 193.66 551.61 ± 39.76 549.76 ± 32.25 198.18 ± 4.62 13,896.86 ± 1586.80 25,480.02

887.16 ± 63.58 5159.22 ± 68.31 – – 804.98 ± 102.48 120.64 ± 20.64 728.69 ± 27.79 194.27 ± 33.13 17,735.58 ± 1133.23 25,630.54

94.98 ± 12.45 433.88 ± 43.54 – – 44.87 ± 8.56 – 304.28 ± 23.85 35.34 ± 6.56 1928.14 ± 301.64 2841.49

– 491.35 ± 44.5 73.74 ± 5.48 313.48 ± 13.72 6910.64 ± 95.56 267.59 ± 18.99 – – – 8056.8

Acids Acetic acid 2-Methyl-butanoic acid ∑

1135.74 ± 147.68 229.72 ± 23.48 1365.46

2217.07 ± 228.49 357.87 ± 55.30 2574.94

1386.03 ± 100.92 467.30 ± 54.97 1853.33

1367.27 ± 154.94 548.17 ± 88.64 1915.44

1094.57 ± 60.09 412.69 ± 59.91 1507.26

83.96 ± 11.82 61.00 ± 5.45 144.96

433.74 ± 28.04 – 433.74

Aldehydes and ketones Acetaldehyde 3,4-Dimethyl-benzaldehyde 3-Octanone 3-Hydroxy-2-butanone ∑ Total

537.12 ± 83.83 78.14 ± 8.02 351.91 ± 25.99 775.27 ± 91.89 1742.44 38,682.81

489.92 ± 79.18 135.54 ± 20.74 788.72 ± 114.88 1857.00 ± 262.22 3271.18 76,766.21

274.87 ± 31.80 205.29 ± 13.03 544.48 ± 3.08 1107.33 ± 34.04 2131.97 51,949.79

188.27 ± 23.34 72.29 ± 7.10 298.51 ± 35.86 1291.85 ± 151.26 1850.92 56,274.96

515.36 ± 46.38 154.24 ± 27.51 53.33 ± 7.31 410.66 ± 3.61 1133.59 65,080.8

16.42 ± 3.67 15.96 ± 2.56 – 239.01 ± 47.45 271.39 6082

– – 1144.95 ± 6.87 – 1144.95 9865.41

a

–: not detected.

Q. Wu et al. / International Journal of Food Microbiology 200 (2015) 39–46

Compounds

Q. Wu et al. / International Journal of Food Microbiology 200 (2015) 39–46

Two acids, two aldehydes, and two ketones were identified in the cultures. S. cerevisiae produced all compounds except for 3-octanone, while A. oryzae only produced acetic acid and 3-octanone. The contents of acids, aldehydes, and ketones observed in co-culture fermentations were higher than those in single culture fermentations, indicating that SSF is an efficient process for production of flavor compounds. 3.5. Statistical analysis of flavor compounds PCA was conducted to gain insight into the nature of the multivariate data and to evaluate biological interactions. Because A. oryzae produced few types of flavor compounds in single culture fermentation, the data for A. oryzae was not considered in the later PCA. As shown in Fig. 4, the experimental groups of single and co-culture fermentations could be distinguished by their metabolic differences. The resulting PCA accounted for 85.32% of the total variance for the first two principal components, with PC1 accounting for 64.38%. Samples from single culture of S. cerevisiae and from the inoculum ratio of 1:0.1 were in the negative phase of PC1, because there were no compounds in this phase, indicating the two samples were correlated with few flavor compounds. Although samples from the inoculum ratios of 1:1 and 1:5 were in the positive phase of PC1, they were near the zero point, indicating that they also correlated with few flavor compounds. Samples from inoculum ratios of 1:0.5 and 1:10 were in the different phase of PC2, indicating the two samples were significantly different in flavor profiles. We found that ethyl nonanoate, ethyl 2-phenylacetate, ethyl 3-phenylpropanoate, ethyl palmitate, ethyl oleate, 2,3-butanediol, 2-phenethyl alcohol, and ethyl 2-methylbutyrate, which were in the positive phase of PC2, were strongly correlated with the inoculum ratio of 1:10, while most of the other compounds were related with

45

the ratio of 1:0.5. This result indicated that SSF was beneficial for flavor production, and the flavor compound metabolism was strongly influenced by the different ratios of S. cerevisiae and A. oryzae. We found that the two ratios of 1:0.5 and 1:10 were both suitable for flavor metabolism, although the flavor metabolism was different. A. oryzae acted not only as a saccharifying agent, but also as a flavor producer. Although it produced fewer varieties of flavor compounds than S. cerevisiae, its second highest production of alcohol occurred at the inoculum ratio of 1:10. Because A. oryzae is an effective alcohol producer, it is likely to contribute significantly to alcohol production at this ratio. In addition, the amount of esters produced was also the most at this ratio. Considering that A. oryzae can efficiently produce acetic acid, the increased ester content might be due to esterification of alcohols and acetic acid. 4. Conclusion SSF is one of the main processes used for production of many types of traditional fermented foods and beverages in Asian countries. In the SSF process, S. cerevisiae grew very effectively, with a maximum biomass that was more than twofold that in the SHF process. The organism also exhibited a higher maximum specific production rate and longer fermentation period for ethanol production, and produced 3.9–7.8 times more combined flavor compounds. In the SHF process, the high initial sugar content led to the early death of S. cerevisiae, and thereby ceased ethanol production. In SSF, a suitable sugar concentration was able to be maintained to allow gradual increases in S. cerevisiae growth and ethanol production. In investigating the interaction between S. cerevisiae and A. oryzae, we found that A. oryzae not only regulated starch saccharification and

Fig. 4. Biplot of the principal components analysis (PC 1 vs. PC 2) of metabolite profiles produced in different SSF and SHF processes. Control, SHF process; A-0.1, ratio of 1:0.1; A-0.5, ratio of 1:0.5; A-1, ratio of 1:1; A-5, ratio of 1:5; A-10, ratio of 1:10.

46

Q. Wu et al. / International Journal of Food Microbiology 200 (2015) 39–46

provided fermentable sugar for S. cerevisiae, but also competed against S. cerevisiae. It is apparent that the two effects combine to regulate cell growth and metabolism, which is important for liquor fermentation. In optimizing the inoculation ratio of S. cerevisiae and A. oryzae, the ethanol yield was the highest for the ratios of 1:0.5, 1:1, and 1:5, and the total amount of flavor compounds was the highest for the ratio of 1:0.5. This implied that the cell growth and metabolic activity of S. cerevisiae could be regulated by the inoculation ratio of A. oryzae in the SSF process by regulating their interaction. The results of this study extend understanding of the cooperation between A. oryzae and S. cerevisiae in co-culture fermentation for Chinese liquor making. The findings outlined here will provide an improved ability to develop and control food and beverage fermentation processes. Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2013AA102108, 2012AA021301), National Natural Science Foundation of China (31371822, 31271921), China Postdoctoral Science Foundation (2014M550265), Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program. References Chen, B., Wu, Q., Xu, Y., 2014. Filamentous fungal diversity and community structure associated with the solid state fermentation of Chinese Maotai-flavor liquor. Int. J. Food Microbiol. 179, 80–84. Fan, W., Qian, M.C., 2006. Characterization of aroma compounds of Chinese “Wuliangye” and “Jiannanchun” liquors by aroma extract dilution analysis. J. Agric. Food Chem. 54, 2695–2704. Farías, M.E., Sosa, O.A., Mendoza, L.M., Fernández, P.A.A., 2010. Microbial Interaction in Fermented Beverages. Nova Publishers, New York. Farid, M.A., El-Enshasy, H.A., Noor El-Deen, A.M., 2002. Alcohol production from starch by mixed cultures of Aspergillus awamori and immobilized Saccharomyces cerevisiae at different agitation speeds. J. Basic Microbiol. 42, 162–171. Furukawa, S., Watanabe, T., Toyama, H., Morinaga, Y., 2013. Significance of microbial symbiotic coexistence in traditional fermentation. J. Biosci. Bioeng. 116, 533–539. Ge, X.Y., Qian, H., Zhang, W.G., 2013. Influence of mixed culture system on the growth performance of Aspergillus oryzae and Saccharomyces cerevisiae. Afr. J. Biotechnol. 12, 3272–3277. Lage, P., Barbosa, C., Mateus, B., Vasconcelos, I., 2014. H. guilliermondii impacts growth kinetics and metabolic activity of S. cerevisiae: the role of initial nitrogen concentration. Int. J Food Microbiol. 172, 62–69.

Lee, W.S., Chen, I.C., Chang, C.H., Yang, S.S., 2012. Bioethanol production from sweet potato by co-immobilization of saccharolytic molds and Saccharomyces cerevisiae. Renew. Energy 39, 216–222. Li, H., Jiao, A., Xu, X., Wu, C., Wei, B., Hu, X., Jin, Z., Tian, Y., 2013. Simultaneous saccharification and fermentation of broken rice: an enzymatic extrusion liquefaction pretreatment for Chinese rice wine production. Bioprocess Biosyst. Eng. 36, 1141–1148. Lin, Y., Tanaka, S., 2006. Ethanol fermentation from biomass resources: current state and prospects. Appl. Microbiol. Biotechnol. 69, 627–642. Lv, X.C., Huang, Z.Q., Zhang, W., Rao, P.F., Ni, L., 2012. Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing. J. Gen. Appl. Microbiol. 58, 33–42. Medina, K., Boido, E., Fariña, L., Gioia, O., Gomez, M.E., Barquet, M., Gaggero, C., Dellacassa, E., Carrau, F., 2013. Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineas. Food Chem. 141, 2513–2521. Morrall, P., Boyd, H.K., Taylor, J.R.N., Van der Walt, W.H., 1986. Effect of germination time, temperature and moisture on malting of sorghum. J. Inst. Brew. 92, 439–445. Murado, M.A., Pastrana, L., Vázquez, J.A., Mirón, J., González, M.P., 2008. Alcoholic chestnut fermentation in mixed culture. Compatibility criteria between Aspergillus oryzae and Saccharomyces cerevisiae strains. Bioresour. Technol. 99, 7255–7263. Nahar, S., Hossain, F., Feroza, B., Halim, M.A., 2008. Production of glucoamylase by Rhizopus sp. in liquid culture. J. Bot. 40, 1693–1698. Olofsson, K., Bertilsson, M., Lidén, G., 2008. A short review on SSF — an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol. Biofuels 1, 7. Sardiñas, N., Vázquez, C., Gil-Serna, J., González-Jaén, M.T., Patiño, B., 2011. Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR. Int. J. Food Microbiol. 145, 121–125. Shin, C.S., Kim, H.J., Kim, M.J., Ju, J.Y., 1998. Morphological change and enhanced pigment production of Monascus when cocultured with Saccharomyces cerevisiae or Aspergillus oryzae. Biotechnol. Bioeng. 59, 576–581. Slivinski, C.T., Machado, A.V.L., Iulek, J., Ayub, R.A., Almeida, M.M.d., 2011. Biochemical characterisation of a glucoamylase from Aspergillus niger produced by solid-state fermentation. Braz. Arch. Biol. Technol. 54, 559–568. Van Beek, S., Priest, F.G., 2002. Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study. Appl. Environ. Microbiol. 68, 297–305. Wang, Y., Yuan, B., Ji, Y., Li, H., 2013. Hydrolysis of hemicellulose to produce fermentable monosaccharides by plasma acid. Carbohydr. Polym. 97, 518–522. Wu, X., Jampala, B., Robbins, A., Hays, D., Yan, S., Xu, F., Rooney, W., Peterson, G., Shi, Y., Wang, D., 2010. Ethanol fermentation performance of grain sorghums (Sorghum bicolor) with modified endosperm matrices. J. Agric. Food Chem. 58, 9556–9562. Wu, Q., Chen, L., Xu, Y., 2013. Yeast community associated with the solid state fermentation of traditional Chinese Maotai-flavor liquor. Int. J. Food Microbiol. 166, 323–330. Wu, Q., Ling, J., Xu, Y., 2014. Starter culture selection for making Chinese sesame-flavored liquor based on microbial metabolic activity in mixed-culture fermentation. Appl. Environ. Microbiol. 80, 4450–4459. Yang, S., Choi, S.J., Kwak, J., Kim, K., Seo, M., Moon, T.W., Lee, Y.W., 2013. Aspergillus oryzae strains isolated from traditional Korean nuruk: fermentation properties and influence on rice wine quality. Food Sci. Biotechnol. 22, 425–432. Zhou, J.M., Ge, X.Y., Zhang, W.G., 2011. Improvement of polygalacturonase production at high temperature by mixed culture of Aspergillus niger and Saccharomyces cerevisiae. Bioresour. Technol. 102, 10085–10088.