The results showed that when the salt concentration was 20%, the suplemented water was 200% (of the raw material) and the fermentation time was 60 days, the contents of amino acids, reducing sugars, total acid were the highest (11.73; 9.87; 8.71 g/100 g dry matter, respectively) and the color of fermented oyster mushroom sauce was the darkest.
AGU International Journal of Sciences – 2019, Vol (3), 57 – 64 EFFECT OF SUPPLEMENTED BRINE CONCENTRATION AND AMOUNT AND FERMENTATION TIME ON OYSTER MUSHROOM (PLEUROTUS SPP.) SAUCE QUALITY Nguyen Thi Ngoc Giang1, Tran Van Khai1 An Giang University, VNU - HCM Information: Received: 09/10/2018 Accepted: 03/01/2019 Published: 11/2019 Keywords: Oyster mushroom, soy sauce, salt concentration, water ratio ABSTRACT Soy sauce has been widely used as one of the main seasoning agents in Asian countries The chemical composition of sauce varies according to the ingredients, preparation attractive and the method of production Sauce is considered delicious when it has color, aroma and the sweetness of protein and sugar The study was investigated the effect of salt concentrations (10, 15, 20, 25% w/v), the amount of water (100, 150, 200, 250, 300% compared with the raw material) and the fermentation time (0, 10, 20, 30, 40, 50, 60, 75, 90 days) to nutrient content as well as color of fermented oyster mushroom sauce The results showed that when the salt concentration was 20%, the suplemented water was 200% (of the raw material) and the fermentation time was 60 days, the contents of amino acids, reducing sugars, total acid were the highest (11.73; 9.87; 8.71 g/100 g dry matter, respectively) and the color of fermented oyster mushroom sauce was the darkest Pleurotus spp., accounting for 46.6-84.8% of dry basis (compared to 60% of A.bisporus) According to Bano and Rajarthnam (1982), carbohydrates consist of 4.2% soluble carbohydrate, 1.7% pentosan and 32.3% hexosan INTRODUCTION Pleurotus spp can be called oyster, abalone or tree mushrooms Pleurotus mushrooms are developed in tropical and subtropical regions and easily artificially cultivated (Akindahunsi and Oyetayo, 2006) Oyster mushrooms have aromatic and flavor properties (Herndndez et al., (2003); Kalmis et al., (2008)) Their nutritional value includes carbohydrates and proteins, which are the main components, accounting for 70 to 90% of dry matter) Besides, they have about 10% ash with many kinds of minerals Fat content is low (1-2%) (Le Xuan Tham, 2010) Proteins of Pleurotus mushrooms are present in all of essential amino acids (Chang and Quimio, 1982) Carbohydrates are the main component of Fermentation is the process of using enzymes of molds to hydrolyze proteins in raw materials into soy sauce The difference between fermentation and rotting is that after fermentation, the product does not produce a rotten smell (Bui Duc Chi Thien, 2014) Fermentation is also an important stage If the raw materials are treated well and mold is grown properly, but the fermentation is not guaranteed, the soy sauce is still unsatisfactory and the efficiency is not high (Nguyen Duc 57 AGU International Journal of Sciences – 2019, Vol (3), 57 – 64 Luong, 2006) In soy sauce production, after raising the mold, the mold is mixed with salt solution In salt solution, the enzymes in the mixture become active The primary biochemical nature in the production of soy sauce is the hydrolysis of starches and proteins by the corresponding amylase and protease enzymes produced by microorganisms The scientific basis of the process is to use the enzyme amylase, protease from microorganisms to break down the starches into sugars with low molecular and proteolysis into polypeptides, peptides, amino acids There is also the formation of alcohol, organic acids All of these compounds create a unique flavor and nutritional value for soy sauce (Nguyen Duc Luong, 2006) study was to determine the brine supplementation and amount as well as the fermentation time on oyster mushroom sauce to reach the best nutritional content and color Factors that affect moromi fermentation are: 2.2 Preparartion of Koji + The addition of salt with high concentration in the fermentation is a necessary condition to hinder the development of undesirable bioburden types However, if the salt concentration is too high, it directly affects the activities of the enzyme systems in the fermentation solution 200 g of oyster mushrooms were steamed until the mushrooms had just reached 90oC and mixed 10% wheat flour The mixture was adjusted to pH6.0 (Jian Chen and Yang Zhu (2013); Perrin (1974)) and added 0.03% mold The koji was incubated at 30oC for 30 hours (Nguyen Thi Ngoc Giang and Nguyen Minh Thuy, 2016; Nguyen Thi Ngoc Giang and Tran Van Khai, 2018) MATERIALS AND EXPERIMENTAL 2.1 Materials Pleurotus mushrooms purchased from Experimental – Practical Area of An Giang University, Viet Nam Fresh mushrooms were washed with water, drained, cut into small spieces (0.5 x cm) and used immediately Aspergillus oryzae molds were provide by the Institute of Biotechnology, Can Tho University, Viet Nam Meizan flour is roasted at 80oC for 15 minutes + The water supplemention has a strong influence on the end product quality Lots of water addition leads to the concentration of nutrients decrease in fermentation sauce If water supplemention is little, the enzyme activities in sauce mass decreases, leading to a reduction in quality, and the cost of the product is increased due to low recovery efficiency (Dang Hong Anh, 2011) Therefore, the choice of supplemented brine concentration and amount is an important condition in soy sauce fermentation 2.3 The experimental design The experiment was designed with three factors and three replicates, including (i): Salt concentration: 10, 15, 20, 25% w/v; (ii) Brine content: 100, 150, 200, 250, 300% compared to the raw material; (iii) Fermentation time: from to 90 days 2.4 Determination of amino acids content Amino acids content was analysed by formol titration (Le Thanh Mai et al., 2005) Amino acids content (mg) is calculated by the formula: + Fermentation time: depending on the amount of water mixed and the fermention temperature, the fermentation time is long or short The fermentation time can also be based on the increase or decrease of formol protein mgN = (A – B) x 1.4 Where, A- volume of NaOH 0.1N used for titration of the sample (mL) The hydrolytic capacity of enzyme system in mold is determined by the content of reducing sugar, amino acids and total acid The present B- volume of NaOH 0.1N used for titration of the blank (mL) 2.5 Determination of reducing sugar content 58 AGU International Journal of Sciences – 2019, Vol (3), 57 – 64 Reducing sugar content was measured by DNS method (Le Thanh Mai et al., 2005) The concentration of reducing sugar was based on standard curve of glucose, y = 23885x + 0.126 , where R2 = 0.9923 L* indicates lightness, a* is the red/green coordinate and b* is the yellow/blue coordinate The difference is calculated: ∆L = L* - Lref (+ = lighter; - = darker) ∆b = b* - bref (+ = yellower; - = bluer) 2.6 Determination of total acid content RESULTS AND DISCUSSIONS Total acid content was determined by titration (Le Thanh Mai et al., 2005) 3.1 Amino acids content (g/100 g dry matter) during fermentation time 2.7 Determination of color differences using L*a*b* (Ngo Anh Tuan, 2006) The effect of salt concentration and brine supplementation on the amino acids content during fermentation time are shown in Table Table Amino acids content (g/100 g dry matter) during fermentation time Salt concentration (%) Brine content (%) 10 Amino acids content (g/100 g dry matter) Fermentation time (days) 10 20 30 40 50 60 75 90 100 3,26 3,41 3,61 6,53 8,55 10,73 8,47 4,38 4,60 10 150 2,94 3,39 4,58 7,20 8,53 10,39 10,28 2,58 1,54 10 200 3,84 1,42 1,24 6,00 8,08 8,81 10,34 7,68 4,27 10 250 2,47 5,27 6,54 8,84 9,05 10,49 11,30 2,47 1,86 10 300 3,02 2,97 4,09 4,90 8,61 10,32 15,37 2,01 1,98 15 100 2,44 3,47 4,75 7,75 7,51 7,91 10,47 2,42 1,55 15 150 3,36 3,69 5,74 6,29 11,00 12,64 8,69 4,91 3,76 15 200 4,00 3,31 7,12 9,48 10,29 11,81 12,09 7,35 5,09 15 250 1,95 3,88 3,73 4,73 7,08 7,83 10,23 4,00 2,21 15 300 2,23 2,73 4,52 4,33 5,70 6,52 10,37 2,46 1,57 20 100 2,65 1,92 4,91 6,12 7,37 8,50 10,29 7,92 3,34 20 150 1,59 3,13 4,23 6,29 7,31 10,14 11,13 11,22 4,32 20 200 3,61 3,68 5,93 6,99 9,16 10,26 11,73 9,18 6,03 20 250 2,71 1,56 4,97 5,89 6,19 10,24 9,31 5,37 3,69 20 300 2,31 1,83 3,10 4,40 5,30 9,25 10,74 5,17 1,05 25 100 2,31 2,17 2,26 2,07 2,98 3,86 7,63 2,45 0,83 25 150 2,45 1,51 2,29 2,86 7,43 8,29 6,59 3,26 2,09 25 200 2,84 3,81 4,87 5,92 8,30 9,96 9,55 6,65 2,15 25 250 1,88 2,33 2,25 2,05 7,21 3,27 5,29 0,89 0,37 25 300 1,93 2,24 2,09 1,87 2,31 7,28 4,20 2,24 0,48 59 AGU International Journal of Sciences – 2019, Vol (3), 57 – 64 The experimental data in Table shows that the amino protein content was high at 10, 15 and 20% salt concentrations (the difference is not statistically significant at 5%) and the lowest at 25% salt concentration According to Le Van Viet Man and Tran Thi Anh Tuyet (2006), the high salt concentration in the sauce (25% by weight) reduced the catalytic activity of protease enzyme In addition, the amount of amino acids was low at high levels of brine supplementation (250 and 300%) or low (100 and 150%) This is also proved similarly in the research of Dang Hong Anh (2011) that solid or dilute masses all affected to the hydrolytic capacity of protease enzymes Amino acids content increased with fermentation time and reached the highest value at day of 60; and then the amount of amino acids gradually decreased Reduction of amino acids value was dued to Maillard reaction The highest amino protein production (11.73 g/100 g dry matter) at 20% salt concentration, 200% brine addition (compared to the weight of raw materials) was on the 60th day of fermentation 3.2 Reducing sugar content (g/100 g dry matter) during fermentation time The effect of salt concentration and brine supplementation on the reducing sugar content during fermentation time are shown in Table Table Reducing sugar content (g/100 g dry matter) during fermentation time Reducing sugar content (g/100 g dry matter) Salt concentration (%) Brine content (%) 10 20 30 40 50 60 75 90 10 100 0,03 0,27 0,40 0,49 0,51 1,34 1,93 0,00 0,00 10 150 0,19 0,79 0,63 1,13 1,42 1,24 3,09 1,49 0,14 10 200 0,05 0,59 1,46 1,65 2,09 2,82 6,37 5,13 0,31 10 250 0,00 0,39 0,57 0,23 0,36 0,52 1,27 0,00 0,00 10 300 0,00 0,18 0,51 0,46 0,64 5,49 5,41 0,46 0,00 15 100 0,05 0,29 0,40 4,03 1,28 1,40 0,37 0,00 0,00 15 150 0,22 0,77 0,76 0,79 1,62 2,34 5,79 0,78 0,41 15 200 0,11 0,37 0,62 2,08 4,22 4,58 0,73 0,00 0,00 15 250 0,00 0,45 1,26 1,44 1,74 7,78 0,34 0,00 0,00 15 300 0,01 0,40 0,31 2,07 2,63 1,23 0,76 0,66 0,00 20 100 0,05 1,69 1,95 2,99 4,17 6,37 7,43 5,90 0,56 20 150 0,58 2,20 2,70 3,22 5,34 5,19 8,,52 5,51 5,93 20 200 0,14 0,65 4,96 4,02 5,47 6,83 9,87 7,67 6,44 20 250 0,03 0,92 1,34 1,41 4,06 5,90 7,78 6,39 0,27 20 300 0,04 0,34 1,45 1,16 1,31 1,73 5,55 3,89 0,37 25 100 0,22 0,29 2,39 2,99 3,62 3,90 9,27 1,07 0,75 25 150 0,19 0,60 1,23 1,88 3,36 4,10 1,48 0,87 0,30 25 200 0,16 0,33 2,49 3,04 5,68 7,08 9,28 5,52 1,96 25 250 0,02 0,42 2,74 4,97 5,26 8,04 0,64 0,31 0,38 25 300 0,00 0,40 2,31 2,70 3,53 4,84 5,14 5,03 2,28 Fermentation time (days) 60 AGU International Journal of Sciences – 2019, Vol (3), 57 – 64 The analytical results in Table show that the reducing sugar content did not differ significantly between the two salt concentrations of 10 and 15% In addition, the high content of reducing sugars was at the 20% salt concentration and the lowest reducing sugar content was at salt concentration of 25% This result was also consistent with the study of Su et al (2005), Dang Hong Anh (2011) that the content of salt from 10-15% did not affect to the activity of the amylase enzyme in the fermentation sauce At high salt concentration, amylase enzyme activity was inhibited and hydrolysis speed in fermentation solution decreased On the other hand, the brine content of 200% (compared to the raw material) gave the highest content of reducing sugar The research of Dang Hong Anh (2011) also determined that the amount of water addition was times the volume of koji produced The content of reducing sugars increases with fermentation time to an optimal value, then decreases The rapid increase of reducing sugars during fermentation is due to the involvement of the amylase enzyme However, due to the aging process, the enzyme activity of the enzyme amylase is denatured in the salt solution and reduced in activity (Chou and Ling, 1998) The reducing sugars content during aging was decreased by the Maillard reaction The highest reducing sugars (9.87 g/100 g of dry matter) at 20% salt concentration, the added salt water content (% compared to raw materials) is 200% at day 60 of the fermentation process 3.3 Total acid content (g/100 g dry matter) during fermentation time Table Total acid content (g/100 g dry matter) during fermentation time Reducing sugar content (g/100 g dry matter) Salt concentration (%) Brine content (%) 10 20 30 40 50 60 75 90 10 100 2,63 4,35 5,03 2,72 1,92 1,72 1,93 1,58 1,70 10 150 2,81 3,11 3,75 4,44 4,50 4,84 6,06 3,03 2,66 10 200 2,91 3,39 3,28 3,65 4,15 5,08 6,63 2,09 0,83 10 250 2,26 2,22 2,66 3,74 4,08 1,95 1,52 1,03 0,92 10 300 1,26 1,45 1,91 2,51 2,40 3,95 3,38 1,70 1,59 15 100 2,25 2,50 2,33 2,11 3,88 4,78 5,49 1,93 0,92 15 150 3,18 2,70 3,21 3,85 4,02 6,37 6,27 3,51 1,82 15 200 2,28 2,62 3,81 4,07 5,16 6,58 8,06 5,58 4,46 15 250 1,91 2,23 2,88 3,54 3,91 4,84 2,81 2,06 1,11 15 300 2,00 3,30 3,48 1,97 2,44 2,48 2,16 1,95 2,08 20 100 1,85 2,34 2,90 2,91 3,14 6,10 7,52 3,11 0,95 20 150 1,60 2,76 2,76 3,76 6,81 6,76 7,64 5,67 1,50 20 200 4,39 7,10 7,60 8,15 4,51 5,66 8,71 3,08 1,43 20 250 2,51 2,06 2,71 3,36 4,96 4,68 5,00 4,78 4,12 20 300 2,08 3,43 3,51 4,16 5,44 5,10 4,85 5,45 3,88 25 100 1,77 2,44 2,85 1,65 2,08 2,99 1,83 1,18 1,40 25 150 2,04 2,99 3,58 1,73 1,56 2,92 2,37 2,11 1,93 25 200 2,44 2,73 3,16 4,12 4,32 4,79 3,81 3,16 0,87 25 250 1,32 1,74 2,43 2,05 1,29 1,34 1,22 0,74 0,98 25 300 1,50 1,63 1,73 1,26 1,23 1,49 1,33 1,27 1,09 Fermentation time (days) 61 AGU International Journal of Sciences – 2019, Vol (3), 57 – 64 The analytical results in Table show that the total acid content increases with the addition of 10 - 20% salt and 100 - 200% water content At 25% salt concentration and 250-300% water supplementation, the total acid content increased slowly because of decreasing enzyme activity The increase in total acidity was due to the action of enzymes that breaked down proteins into amino acids, peptides (containing carbolylic chains), reducing sugars (Shieh et al., 1982) The highest total acidity produced (8.71 g/100 g dry matter) at a salt concentration of 20% and 200% water addition (compared to the material) on the 60th day of fermentation affects the darkening of soy sauce (Choi et al., 1990) The correlations between the salt concentration and brine supplementation (% compared to the raw material) to color of soy sauce are presented in Figure and Figure As shown in these figures, both of salt concentration and brine supplementation affected to color of the solution at the 60th day of fermentation The darkening of the sauce increased with increasing salt concentration and additional water content Meanwhile, the yellow color of the solution decreased with decreasing salt concentration and brine supplementation Maillard reaction affected to the nutrients (reducing sugars content and amino acids), affected to the color and flavor of the product The browning rate depends on the water content (Le Ngoc Tu, 2002) At the 20% salt concentration, the salt brine content is 200%, the solution has the darkest yellow color (and the highest content of amino acids, reducing sugars) The change in color of soy sauce during fermentation time is shown in Figure 3.4 The change in color of soy sauce during fermentation Color is one of the important parameters to evaluate the effectiveness of the fermentation process The color of soy sauce is formed through two stages: fermentation and pasteurization (Miyagi et al , 2013) This is all due to browning, known as a Maillard reaction between amines and reducing sugars (Yokotsuka, 1986) Melanoidin from the Maillard reaction is the main ingredient that Figure Correlation of color ∆L with salt concentration and brine supplementation (% compared to raw material) Figure Correlation of color ∆b with salt concentration and brine supplementation (% compared to raw material) 62 AGU International Journal of Sciences – 2019, Vol (3), 57 – 64 Figure The change in color of soy sauce during fermentation time mushrooms – Biological nature and cultivation methods Chinese University Press, Hong Kong, pp 363 – 380 CONCLUSION Soy sauce produced by fermentation method almost retains all the amino acids in the material Fermented sauces also contain quite a lot of sugars because of the effect of the enzyme amylase on starch The fermentation of oyster mushroom sauce under the effect of Aspergillus oryzae is carried out in 60 days with an additional salt concentration of 20% and brine content of 200% (compared to the raw material) that will give a high nutrient content of sauce (11,73; 9,87; 8,71 g/100 g dry matter, respectively) and the darkest-yellow Bui Duc Chi Thien (2014) Technology for fermentation of soy sauce 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Reducing sugar content (g/100 g dry matter) during fermentation time The effect of salt concentration and brine supplementation on the reducing sugar content during fermentation time are shown... two salt concentrations of 10 and 15% In addition, the high content of reducing sugars was at the 20% salt concentration and the lowest reducing sugar content was at salt concentration of 25% This... (Nguyen Duc Luong, 2006) study was to determine the brine supplementation and amount as well as the fermentation time on oyster mushroom sauce to reach the best nutritional content and color Factors