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The enhancement and encapsulation of Agaricus bisporus flavor
Journal of Food Engineering 65 (2004) 391–396 www.elsevier.com/locate/jfoodeng
The enhancement and encapsulation of Agaricus bisporus flavor Zhi-qiang Liu a
a,*
, Jian-hong Zhou a, Yun-long Zeng a, Xu-long Ouyang
b
Department of Biology and Environment Engineering, Hunan University of Science and Technology, Xiangtan, 411201 Hunan, China b Department of Biological Engineering, Shaoyang University, Hunan, Shaoyang 422004 Hunan, China Received 24 June 2003; accepted 26 January 2004
Abstract 1-octene-3-ol, the major flavoring substance in Agaricus bisporus, is formed by the reaction of linolenic acid in the presence of enzyme. By adding sunflower oil hydrolysate rich in linolenic acid into the system, we enhanced the flavor formation, then extracted flavors with ethanol while treating them with complex enzymes, later using spray drying to produce a microencapsulated powder flavor. The experiment on flavor enhancement showed that the enhancement effect reached its maximum when the pH is 6.5, temperature is 35 °C and mixed fatty acid is 0.3%.The experiment on the encapsulation of flavor showed the best ratio of the carriers is (calculated according to the weight of fresh mushroom): soybean hydrolyzing protein 10%, Arabic gum 1%, dextrin 15%. The most suitable process was a feed temperature, air inlet and air outlet temperature during spray drying of 50–60, 130–140 and 70– 80 °C respectively. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Agaricus bisporus; 1-octene-3-ol; Flavor enhancement; Encapsulation
1. Introduction Rich in nutrition, low in fat as well as having a special flavor, Agaricus bisporus is well accepted by consumers. The production of A. bisporus in China holds the third place in the world. As mushroom has different kinds of enzymes with highly activity, the fresh mushroom must be processed immediately after picked, otherwise it will quickly lose flavor and quality. The main processing method of mushroom at present, is canning, but the flavor and quality is seriously impaired during the process. Though there is mushroom soup stock for instant noodles on the market, the flavor is unsatisfactory. So the study of flavoring agents with strong mushroom flavor is very important in practice. Cruz, Suberville, and Montury (1997) and Husson, Bompas, Kermasha, and Belin (2001) study showed that 1-octene-3-ol, the main flavor chemical compound in A. bisporus, is produced through the effect of lipoxidase in the cell to produce hydroperoxide with O2 in the air, and then through the effect of hydroperoxide lyase and oxi-
*
Corresponding author. Tel.: +86-732-829-0700; fax: +86-732-8290509. E-mail addresses: [email protected], [email protected] (Z.-q. Liu). 0260-8774/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.01.038
dase and reducase. So linolenic acid is the precursor to produce l-octene-3-ol (Bonzom, Zloh, Baldeo, Gibbons, & Nicolaou, 1999). Tressl, Bahri, and Engel (1980) added 0.03% of linolenic acid to mushroom and kept it heated. The result showed that the content of 1-octene3-ol increased more than three times, which demonstrated the possibility of intensifying mushroom flavor by using the mushroom itself as the enzyme resource when adding linolenic acid as a flavor precursor. As the cost of pure linolenic acid is very high, in the practical production we used a mixed fatty acid hydrolysate from sunflower oil whose content of linolenic acid was more than 50%. Because the flavor compounds in food are mostly volatiles, microcapsulation was applied to produce a powdered flavor in order to prevent the flavor loss, to prolong the product’s shelf life and make it convenient to use (Usha & Pothakamury, 1995).
2. Materials A. bisporus and sunflower oil were obtained from the local market. The pectinase was produced in Aspergillus niger by Nove Nordisk which is a company in Denmark and was
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Z.-q. Liu et al. / Journal of Food Engineering 65 (2004) 391–396
commercially named Pectinex ultra sp-1. This has a declared activity of 26,000 galacturonic acid/ml.
solution added and chilled. The upper oil phase was sunflower oil hydrolysate (mixed fatty acids). 3.1.2. Mushroom flavor enhancement 500 g of clean fresh mushroom were weighed out and then mixed with a 0.1 M buffer solution of phosphate of pH 6.5 in the proportions of 1:1; then some fatty acids were added. All was triturated in a triturator for quarter hour and then once every 20 s. The thick liquid was kept warm for 30 min at a fixed temperature and pH.
3. Experiment method 3.1. Technological process The process is shown in Fig. 1. 3.1.1. The preparation of sunflower oil hydrolysate A solution was prepared by weighing out 20 g of sunflower oil and pouring it into a 500 ml saponification bottle, adding 250 ml of 0.5 mol L1 KOH-alcohol solution and heating it to 90 °C in a thermostatic water bath (with reflux condensation) for one hour until saponification was finished. The mixture was then cooled and the pH adjusted to 3.0 by concentrated H2 SO4 . The acidized hydrolysate was poured into a 1000 ml separating funnel and a little saturated NaCl
3.1.3. Alcohol extraction After the flavour enhancement with the fatty acids, a residue of mushroom was obtained to which we added 0.4% citric acid and 0.05% emulsifying agent (Span 20 and span 80 and mixed them with fatty acid in the ratio of 2:1:1). Immersing it in 80% alcohol solution at a reduced pressure at room temperature for about 10–15 min, we added 0.1% phytic acid, 0.3% sodium metaphosphate and 0.1% vitamin C solution while homo-
Sunflower oil
Mushroom Cleaning and drying
Hydrolysis Mixed fatty acids
Adding buffer solution
Crushing and keeping warm (enhancing flavor)
Centrifugal separatin
Liquid l
Liquid 2
Residue Alcohol extraction residue
Liquid 3
Na2CO3 - NaCl Solution extraction residue
Liquid 4 Concentration Adding ingredient
Concentrate
Enzyme treament and extraction Waste Adding carriers
Homogenization
Spray drying
Fig. 1. Technological process of the enhancement and encapsulation of A. bisporus flavor.
Z.-q. Liu et al. / Journal of Food Engineering 65 (2004) 391–396
genizing at 95 °C for 10 min. This was later separated by centrifugation. 3.1.4. Na2 CO3 –NaCl solution extraction 0.1% Na2 CO3 solution containing 0.3% NaCl was added to the residue which was obtained from the alcohol extraction. The extraction time was 20 min at a temperature of more than 80 °C. Finally centrifugal separation was applied to this mixture. 3.1.5. Enzyme hydrolysis and re-extraction The residue produced in the previous step was dispersed in citric acid buffer solution of pH 4.0 and 0.5% complex pectinase was added. Then the liquid was shaken for 50 min at 40 °C and centrifuged later. 3.1.6. Mushroom flavor capsulation All ingredients involving mushroom extracts and microencapsule carrier materials were completely mixed in a triturator and homogenizing in a JHC homogenizer at 30 MPa for 10 min and then using spray dried to produce a microencapsulated mushroom flavor. 3.2. Quality evaluation of the microencapsulated product For assessment of the capsulation effect (Hong, Yu, Noh, & Chang, 2002), two microcapsule samples, one of dry powder ðS1 Þ, the other dissolved in water ðS2 Þ, were assessed by organoleptic evaluation by 14 people. The flavor intensity of the mixed flavor oil ranged over a scale of five degrees of 0, 1, 2, 3, 4. These had the meanings:- no flavour, some flavour, definite, strong and very strong flavours, respectively. The final result was examined by a t-test to determine whether the two samples were significantly different. The particle size of the microcapsule was determined with a light microscope (Liu, Takuroh, & Furuta, 2001). Flavor enhancement effect assessment or scoring by organoleptic evaluation was adopted to assess flavor strength (Junyaprasert, Mitrevej, Sinchaipanid, Boonme, & Wurster, 2001). The moisture content was determined by loss in weight following the drying of a sample in a vacuum oven at 70 °C for 24 h, as described by the AOAC (1990). The yield of microcapsule was calculated as the following (Shahidi & Han, 1993): The yieldð%Þ ¼
393
such as Glu, 50 -GMP and mycopolysaccharide which is good for health (Wu, Zhang, & Yang, 2003). As 1-octene-3-ol is a colorless oily liquid, insoluble in water but easy to dissolve in alcohol (Hong et al., 2002), ordinary water extraction cannot extract it thoroughly. At the same time, the sporocarp cell wall cannot be destroyed completely, so the extraction rate of polysaccharide and flavor material is very low. In order to solve this problem, two methods were adopted. First, after flavor enhancement, a little 80% alcohol was added to increase the sporocarp pieces, which converted insoluble 1-octene-3-ol into alcohol. Secondly, enzyme treatment was applied at the last leaching step. The study showed that the use of a complex enzyme system which included pectinase, cellulase, and protease to treat the sporocarp could effectively degrade and cause the collapse of the sporocarp cell wall. The result showed that the mushroom flavor was obviously enhanced with these treatments. 4.2. Mushroom flavor enhancement 4.2.1. Single factor experiment The main factors affecting on flavor enhancement are pH, temperature, holding time and the quantity of mixed fatty acids (Table 1). We discussed these factors as follows. 1. Holding time: The influence of heating time on the enhancement effect of mushroom flavor was studied in the system with a pH of 6.0, sunflower oil fatty acid contents of 0.15%, a temperature of 20 °C and a heating time that varied from 0 to 60 min. The experimental results showed that mushroom flavor increased rapidly in the first 40 min but not so obviously after that. Meanwhile, the polyphenoloxidase activity in the mushroom was so strengthened that it caused the mushroom slurry to be brown. Since the reaction time could not be prolonged excessively, the proper enhancement time was taken to be 40 min. 2. Temperature: The influence of temperature on the enhancement effect of mushroom flavor was studied in the system with pH 6.5, sunflower oil fatty acid contents of 0.15%, a heating/holding time of 30 min and a temperature that varied from 20 to 60 °C. The results showed that the flavor strength was kept
Total weight of microcapsuleðgÞ ð1-water contentÞ 100%: Total weight of solid materialðgÞ
4. Results and analysis
Table 1 Factors and levels of orthogonal experiment L9 (34 ) Factor
A (°C) temperature
B (%) fatty acids added
C (pH)
Level 1 Level 2 Level 3
25 30 35
0.3 0.35 0.4
6.0 6.5 7.0
4.1. Technical analysis of flavor extraction The main flavor materials in mushroom flavor extraction are 1-octene-3-ol and other flavor materials
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at a high level as the temperature increased in the range of 20–40 °C but would be weakened if the temperature was higher than 40 °C because of the decline in enzyme activity. 3. System pH: When the system pH varied from 4.0 to 8.0, the experimental results indicated that the flavor strength was higher at pH 6–7. When the system pH was more acidic or more alkaline, the enzyme activity would be affected and the flavor strength would be weakened. 4. Amount of sunflower oil fatty acids: The influence of the amount of sunflower oil fatty acids on the enhancement effect of mushroom flavor was studied in the system with pH 6.5. a heating/holding time of 30 min, a temperature of 20 °C, and an amount of sunflower oil fatty acids that varied from 0.1% to 0.4%. The experimental results showed that the flavor strength increased as the amount of mixed fatty acids added increased, but would only be increased slowly when the amount of fatty acids added exceeded 0.4%. 4.2.2. Results and analysis of orthogonal experiment (Tables 2 and 3) The variance analysis showed that pH and temperature were both significant factors but fatty acids added was not and the preferred selection was pH 6.5 and preferred temperature was 35 °C (Tables 2 and 3). Because the amount of fatty acids added had no significant effect, we chose the lower addition level of 0.3%. 4.3. Encapsulation of flavor extract 4.3.1. Determination of carrier materials and formula Usha and Pothakamury (1995) suggested that a microcapsule carrier material must have a good emulsibility and film-forming ability and have a flat flavor.
Table 2 L9 (34 ) Orthogonal experiment table Test
A
B
C
Flavor score
1 2 3 4 5 6 7 8 9 K1 K2 K3 R
1 1 1 2 2 2 3 3 3 12.21 18.18 22.06 3.92
1 2 3 1 2 3 1 2 3 18.52 17.13 16.76 1.76
1 2 3 2 3 1 3 1 2 14.79 23.47 14.15 9.32
3.23 5.87 3.11 8.78 4.53 4.83 6.51 6.73 8.82
Table 3 Analysis of variance components of results of orthogonal test Source
Sum of square
Freedom degree
F value
Significance
A B C D (error)
16.39 0.58 18.07 0.73
2 2 2 2
22.14 0.70 24.43
(Yes) Not (Yes)
F0:10 ð2:2Þ ¼ 9, F0:05 ð2:2Þ ¼ 19.
At the same time, it must be inexpensive and available. Dextrin, Arabic gum and protein (milk serum, bovine serum albumin, dried defatted soybean powder etc.) usually serve as carrier materials. Considering that a single carrier material has difficulty in meeting the process requirement, the composition and proportion of carrier materials were determined (according to a singlefactor experiment) by the weight of mushroom slurry to be a soybean protein hydrolysate content of 10%, a gum Arabic content of 1% and a dextrin content of 15%.
4.3.2. Spray drying Reineccius (1988) suggested that for a successful microcapsulated product, even if the carrier materials were suitable, the yield of microcapsule product and the encapsulation effect might not be satisfactory if the optimum spray drying conditions were not used. The study indicated that the main factors in the spray drying were feed temperature, air inlet temperature, and air outlet temperature. Feed temperature affected the fluidity of the liquid, the difficulty of spray drying, the viscosity of the product and so on. We could increase the feed temperature to reduce the viscosity of liquid, which reduced the mean size of the droplets. However, the temperature should not be so high that it results in the volatilization of some volatile ingredients. We used a feed temperature of 50– 60 °C. Air inlet temperature has strong connection with the drying velocity and the ability to dry the microcapsule product, to achieve a granular structure with a suitable water content and the stability in the finished microcapsule product. The experiments showed that a suitable air inlet temperature was 130–140 °C. Beatus, Rziel, Rosenberg, and Kopelman (1985) suggested that when the air inlet temperature was low, the evaporation ability was not high enough to form the high density and intensive capsule membrane. At the same time, the product would have a high water content and have such a poor fluidity that it easily adhered to the wall; When the air inlet temperature was high, the excessive evaporation could easily cause a crack in the membrane as well as loss of flavoring ingredients through volatiliza-
Z.-q. Liu et al. / Journal of Food Engineering 65 (2004) 391–396
tion and the decomposition of heat-sensitive component at high temperature. This also causes off-flavors. Air outlet temperature had a significant effect on the product water content and the microencapsulated structure. Air outlet temperature must be controlled with the air inlet temperature. If the air outlet temperature was high, it will help to form an integrated and compact wall structure and thus improve the drying effect. Bhandari, Dumoulin, Richard, Nolean, and Lebert (1992) found that if the air outlet temperature were too high, the product would crack because of overheating. The product was ideal when the air outlet temperature was 70–80 °C.
4.3.3. Characteristics of the product The microencapsulated mushroom flavor product of our experiments was a globular micro granule that was light yellow in color and had a good dispersibility and solubility. Fifty particles selected at random were examined under a microscope. The particle size was about 40–90 lm, averaged at 65 lm; water content of the microcapsule was measured as 3.8%; the yield of microcapsules in our experiments was 92.1%.
395
reason for these was that the flavor was not released unless heated or dissolved in water. 5. Conclusion (1) The preferred technology process to extract mushroom flavor was determined in our study. (2) The mushroom flavor could be strengthened by adding sunflower oil hydrolysate (mixed fatty acids) into the mushroom. The preferred conditions for enhancement were obtained from a single-factor experiment and an orthogonal experiment and selected as follows: amount of sunflower oil mixed fatty acids 0.3%, pH 6.5, heating time 40 min, temperature 35 °C. The flavor of the product was obviously strengthened under these conditions. (3) Spray drying was applied. The ratio of the carriers (calculated as the weight of fresh mushroom) was soybean hydrolyzing protein 10%, Arabic gum 1%, and dextrin 15%. Feed temperature, the air inlet and air outlet temperature during spray drying were 50–60, 130– 140 and 70–80 °C respectively. References
4.3.4. The results of the encapsulation effect as measured by organoleptic evaluation are stated in Table 4 The final result was examined by t-test, the value of t being 12.15. From the t-test tables we saw that t0:01 26 ¼ 2:4786, t0:005 20 ¼ 2:7787, so the value was far higher than the critical value (Table 4). In the evaluation results, the dry powder sample was remarkably different, the score for the dissolved sample being far higher than that of dry powder sample, which showed that the encapsulation effect was obvious to the panel lists. The
Table 4 Scores of encapsulation mushroom flavor product by organoleptic evaluation Judger
Dry powder ðS1 Þ
Dissolved in water ðS2 Þ
Difference
Difference squared
1 2 3 4 5 6 7 8 9 10 11 12 13 14
0 1 1 0 0 1 1 2 0 1 1 1 0 2
3 3 4 4 4 3 3 4 4 4 4 2 3 4
3 2 3 4 4 2 2 4 3 3 1 1 3 2
9 4 9 16 16 4 4 16 9 9 1 1 9 4
AOAC (1990). Official methods of analysis (15th ed). Washington, DC: Association of Official Analytical Chemists. Beatus, Y., Rziel, A., Rosenberg, M., & Kopelman, I. J. (1985). Spray drying microencapsulation of paprika oleoresin. Lebensm. Will-utechnol, (18), 28. Bhandari, B. R., Dumoulin, E. D., Richard, H. M. J., Nolean, I., & Lebert, A. M. (1992). Flavor encapsulation by spray drying, Application to citral and linalyl acetate. Journal of Food Science, 57(1), 217. Bonzom, P., Zloh, M. A., Baldeo, M., Gibbons, W., & Nicolaou, W. A. (1999). NMR lipid profile of agaricus bisporus. Phytochemistry, 50(8), 1311–1321. Cruz, C., Suberville, C., & Montury, M. (1997). Fatty acid content and some flavor compound release in two strains of agaricus bisporus according to three stages of development. Journal of Agricultural and Food Chemistry, 45(1), 64–67. Hong, E.-M., Yu, M.-G., Noh, B.-S., & Chang, P.-S. (2002). Optimization of onion oil microencapsulation by response surface methodology. Korean Journal of Food Science and Technology, 134(3), 437–443. Husson, F., Bompas, D., Kermasha, S., & Belin, J. M. (2001). Biogeneration of 1-octen-3-ol by lipoxygenase and hydroperoxide lyase activities of Agaricus bisporus. Process Biochemistry, 37(2), 177–182. Junyaprasert, V. B., Mitrevej, A., Sinchaipanid, N., Boonme, P., & Wurster, D. E. (2001). Effect of process variables on the microencapsulation of vitamin A palmitateby gelation-acacia coacervation. Drug development and Industrial Pharmacy, 27(6), 561–566. Liu, X.-D., Takuroh, A., & Furuta, T. (2001). Takeshi microencapsulation of emulsified hydrophobic flavors by spray drying. Drying Technology, 19(7), 1361–1374. Reineccius, G. A. (1988). Spray-drying of food flavors in flavor encapsulation. In G. A. Reineccius & S. J. Risch (Eds.), ACS Symposium Series No. 370 (pp. 56–66). Washington, DC: American Chemical Society.
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Shahidi, F., & Han, X.-Q. (1993). Encapsulation of feed ingredients. Critical Reviews in Food Science and Nutrition, 33(6), 501– 547. Tressl, R., Bahri, D., & Engel, K. H. (1980). ACS Symposium Series No. 170 (pp. 213–232). Washington, DC: American Chemical Society.
Usha, R., & Pothakamury, U. R. (1995). Fundamental aspects of controlled release in foods. Trends in Food Science and Technology, 6(12), 397–406. Wu, J.-x., Zhang, H.-y., & Yang, R. (2003). Monosaccharide composition and extraction of the polysaccharide from Agaricus Bisporus fruiting bodies. Edible Fungi of China, 22(1), 31–32.
The enhancement and encapsulation of Agaricus bisporus flavor Zhi-qiang Liu a
a,*
, Jian-hong Zhou a, Yun-long Zeng a, Xu-long Ouyang
b
Department of Biology and Environment Engineering, Hunan University of Science and Technology, Xiangtan, 411201 Hunan, China b Department of Biological Engineering, Shaoyang University, Hunan, Shaoyang 422004 Hunan, China Received 24 June 2003; accepted 26 January 2004
Abstract 1-octene-3-ol, the major flavoring substance in Agaricus bisporus, is formed by the reaction of linolenic acid in the presence of enzyme. By adding sunflower oil hydrolysate rich in linolenic acid into the system, we enhanced the flavor formation, then extracted flavors with ethanol while treating them with complex enzymes, later using spray drying to produce a microencapsulated powder flavor. The experiment on flavor enhancement showed that the enhancement effect reached its maximum when the pH is 6.5, temperature is 35 °C and mixed fatty acid is 0.3%.The experiment on the encapsulation of flavor showed the best ratio of the carriers is (calculated according to the weight of fresh mushroom): soybean hydrolyzing protein 10%, Arabic gum 1%, dextrin 15%. The most suitable process was a feed temperature, air inlet and air outlet temperature during spray drying of 50–60, 130–140 and 70– 80 °C respectively. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Agaricus bisporus; 1-octene-3-ol; Flavor enhancement; Encapsulation
1. Introduction Rich in nutrition, low in fat as well as having a special flavor, Agaricus bisporus is well accepted by consumers. The production of A. bisporus in China holds the third place in the world. As mushroom has different kinds of enzymes with highly activity, the fresh mushroom must be processed immediately after picked, otherwise it will quickly lose flavor and quality. The main processing method of mushroom at present, is canning, but the flavor and quality is seriously impaired during the process. Though there is mushroom soup stock for instant noodles on the market, the flavor is unsatisfactory. So the study of flavoring agents with strong mushroom flavor is very important in practice. Cruz, Suberville, and Montury (1997) and Husson, Bompas, Kermasha, and Belin (2001) study showed that 1-octene-3-ol, the main flavor chemical compound in A. bisporus, is produced through the effect of lipoxidase in the cell to produce hydroperoxide with O2 in the air, and then through the effect of hydroperoxide lyase and oxi-
*
Corresponding author. Tel.: +86-732-829-0700; fax: +86-732-8290509. E-mail addresses: [email protected], [email protected] (Z.-q. Liu). 0260-8774/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.01.038
dase and reducase. So linolenic acid is the precursor to produce l-octene-3-ol (Bonzom, Zloh, Baldeo, Gibbons, & Nicolaou, 1999). Tressl, Bahri, and Engel (1980) added 0.03% of linolenic acid to mushroom and kept it heated. The result showed that the content of 1-octene3-ol increased more than three times, which demonstrated the possibility of intensifying mushroom flavor by using the mushroom itself as the enzyme resource when adding linolenic acid as a flavor precursor. As the cost of pure linolenic acid is very high, in the practical production we used a mixed fatty acid hydrolysate from sunflower oil whose content of linolenic acid was more than 50%. Because the flavor compounds in food are mostly volatiles, microcapsulation was applied to produce a powdered flavor in order to prevent the flavor loss, to prolong the product’s shelf life and make it convenient to use (Usha & Pothakamury, 1995).
2. Materials A. bisporus and sunflower oil were obtained from the local market. The pectinase was produced in Aspergillus niger by Nove Nordisk which is a company in Denmark and was
392
Z.-q. Liu et al. / Journal of Food Engineering 65 (2004) 391–396
commercially named Pectinex ultra sp-1. This has a declared activity of 26,000 galacturonic acid/ml.
solution added and chilled. The upper oil phase was sunflower oil hydrolysate (mixed fatty acids). 3.1.2. Mushroom flavor enhancement 500 g of clean fresh mushroom were weighed out and then mixed with a 0.1 M buffer solution of phosphate of pH 6.5 in the proportions of 1:1; then some fatty acids were added. All was triturated in a triturator for quarter hour and then once every 20 s. The thick liquid was kept warm for 30 min at a fixed temperature and pH.
3. Experiment method 3.1. Technological process The process is shown in Fig. 1. 3.1.1. The preparation of sunflower oil hydrolysate A solution was prepared by weighing out 20 g of sunflower oil and pouring it into a 500 ml saponification bottle, adding 250 ml of 0.5 mol L1 KOH-alcohol solution and heating it to 90 °C in a thermostatic water bath (with reflux condensation) for one hour until saponification was finished. The mixture was then cooled and the pH adjusted to 3.0 by concentrated H2 SO4 . The acidized hydrolysate was poured into a 1000 ml separating funnel and a little saturated NaCl
3.1.3. Alcohol extraction After the flavour enhancement with the fatty acids, a residue of mushroom was obtained to which we added 0.4% citric acid and 0.05% emulsifying agent (Span 20 and span 80 and mixed them with fatty acid in the ratio of 2:1:1). Immersing it in 80% alcohol solution at a reduced pressure at room temperature for about 10–15 min, we added 0.1% phytic acid, 0.3% sodium metaphosphate and 0.1% vitamin C solution while homo-
Sunflower oil
Mushroom Cleaning and drying
Hydrolysis Mixed fatty acids
Adding buffer solution
Crushing and keeping warm (enhancing flavor)
Centrifugal separatin
Liquid l
Liquid 2
Residue Alcohol extraction residue
Liquid 3
Na2CO3 - NaCl Solution extraction residue
Liquid 4 Concentration Adding ingredient
Concentrate
Enzyme treament and extraction Waste Adding carriers
Homogenization
Spray drying
Fig. 1. Technological process of the enhancement and encapsulation of A. bisporus flavor.
Z.-q. Liu et al. / Journal of Food Engineering 65 (2004) 391–396
genizing at 95 °C for 10 min. This was later separated by centrifugation. 3.1.4. Na2 CO3 –NaCl solution extraction 0.1% Na2 CO3 solution containing 0.3% NaCl was added to the residue which was obtained from the alcohol extraction. The extraction time was 20 min at a temperature of more than 80 °C. Finally centrifugal separation was applied to this mixture. 3.1.5. Enzyme hydrolysis and re-extraction The residue produced in the previous step was dispersed in citric acid buffer solution of pH 4.0 and 0.5% complex pectinase was added. Then the liquid was shaken for 50 min at 40 °C and centrifuged later. 3.1.6. Mushroom flavor capsulation All ingredients involving mushroom extracts and microencapsule carrier materials were completely mixed in a triturator and homogenizing in a JHC homogenizer at 30 MPa for 10 min and then using spray dried to produce a microencapsulated mushroom flavor. 3.2. Quality evaluation of the microencapsulated product For assessment of the capsulation effect (Hong, Yu, Noh, & Chang, 2002), two microcapsule samples, one of dry powder ðS1 Þ, the other dissolved in water ðS2 Þ, were assessed by organoleptic evaluation by 14 people. The flavor intensity of the mixed flavor oil ranged over a scale of five degrees of 0, 1, 2, 3, 4. These had the meanings:- no flavour, some flavour, definite, strong and very strong flavours, respectively. The final result was examined by a t-test to determine whether the two samples were significantly different. The particle size of the microcapsule was determined with a light microscope (Liu, Takuroh, & Furuta, 2001). Flavor enhancement effect assessment or scoring by organoleptic evaluation was adopted to assess flavor strength (Junyaprasert, Mitrevej, Sinchaipanid, Boonme, & Wurster, 2001). The moisture content was determined by loss in weight following the drying of a sample in a vacuum oven at 70 °C for 24 h, as described by the AOAC (1990). The yield of microcapsule was calculated as the following (Shahidi & Han, 1993): The yieldð%Þ ¼
393
such as Glu, 50 -GMP and mycopolysaccharide which is good for health (Wu, Zhang, & Yang, 2003). As 1-octene-3-ol is a colorless oily liquid, insoluble in water but easy to dissolve in alcohol (Hong et al., 2002), ordinary water extraction cannot extract it thoroughly. At the same time, the sporocarp cell wall cannot be destroyed completely, so the extraction rate of polysaccharide and flavor material is very low. In order to solve this problem, two methods were adopted. First, after flavor enhancement, a little 80% alcohol was added to increase the sporocarp pieces, which converted insoluble 1-octene-3-ol into alcohol. Secondly, enzyme treatment was applied at the last leaching step. The study showed that the use of a complex enzyme system which included pectinase, cellulase, and protease to treat the sporocarp could effectively degrade and cause the collapse of the sporocarp cell wall. The result showed that the mushroom flavor was obviously enhanced with these treatments. 4.2. Mushroom flavor enhancement 4.2.1. Single factor experiment The main factors affecting on flavor enhancement are pH, temperature, holding time and the quantity of mixed fatty acids (Table 1). We discussed these factors as follows. 1. Holding time: The influence of heating time on the enhancement effect of mushroom flavor was studied in the system with a pH of 6.0, sunflower oil fatty acid contents of 0.15%, a temperature of 20 °C and a heating time that varied from 0 to 60 min. The experimental results showed that mushroom flavor increased rapidly in the first 40 min but not so obviously after that. Meanwhile, the polyphenoloxidase activity in the mushroom was so strengthened that it caused the mushroom slurry to be brown. Since the reaction time could not be prolonged excessively, the proper enhancement time was taken to be 40 min. 2. Temperature: The influence of temperature on the enhancement effect of mushroom flavor was studied in the system with pH 6.5, sunflower oil fatty acid contents of 0.15%, a heating/holding time of 30 min and a temperature that varied from 20 to 60 °C. The results showed that the flavor strength was kept
Total weight of microcapsuleðgÞ ð1-water contentÞ 100%: Total weight of solid materialðgÞ
4. Results and analysis
Table 1 Factors and levels of orthogonal experiment L9 (34 ) Factor
A (°C) temperature
B (%) fatty acids added
C (pH)
Level 1 Level 2 Level 3
25 30 35
0.3 0.35 0.4
6.0 6.5 7.0
4.1. Technical analysis of flavor extraction The main flavor materials in mushroom flavor extraction are 1-octene-3-ol and other flavor materials
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at a high level as the temperature increased in the range of 20–40 °C but would be weakened if the temperature was higher than 40 °C because of the decline in enzyme activity. 3. System pH: When the system pH varied from 4.0 to 8.0, the experimental results indicated that the flavor strength was higher at pH 6–7. When the system pH was more acidic or more alkaline, the enzyme activity would be affected and the flavor strength would be weakened. 4. Amount of sunflower oil fatty acids: The influence of the amount of sunflower oil fatty acids on the enhancement effect of mushroom flavor was studied in the system with pH 6.5. a heating/holding time of 30 min, a temperature of 20 °C, and an amount of sunflower oil fatty acids that varied from 0.1% to 0.4%. The experimental results showed that the flavor strength increased as the amount of mixed fatty acids added increased, but would only be increased slowly when the amount of fatty acids added exceeded 0.4%. 4.2.2. Results and analysis of orthogonal experiment (Tables 2 and 3) The variance analysis showed that pH and temperature were both significant factors but fatty acids added was not and the preferred selection was pH 6.5 and preferred temperature was 35 °C (Tables 2 and 3). Because the amount of fatty acids added had no significant effect, we chose the lower addition level of 0.3%. 4.3. Encapsulation of flavor extract 4.3.1. Determination of carrier materials and formula Usha and Pothakamury (1995) suggested that a microcapsule carrier material must have a good emulsibility and film-forming ability and have a flat flavor.
Table 2 L9 (34 ) Orthogonal experiment table Test
A
B
C
Flavor score
1 2 3 4 5 6 7 8 9 K1 K2 K3 R
1 1 1 2 2 2 3 3 3 12.21 18.18 22.06 3.92
1 2 3 1 2 3 1 2 3 18.52 17.13 16.76 1.76
1 2 3 2 3 1 3 1 2 14.79 23.47 14.15 9.32
3.23 5.87 3.11 8.78 4.53 4.83 6.51 6.73 8.82
Table 3 Analysis of variance components of results of orthogonal test Source
Sum of square
Freedom degree
F value
Significance
A B C D (error)
16.39 0.58 18.07 0.73
2 2 2 2
22.14 0.70 24.43
(Yes) Not (Yes)
F0:10 ð2:2Þ ¼ 9, F0:05 ð2:2Þ ¼ 19.
At the same time, it must be inexpensive and available. Dextrin, Arabic gum and protein (milk serum, bovine serum albumin, dried defatted soybean powder etc.) usually serve as carrier materials. Considering that a single carrier material has difficulty in meeting the process requirement, the composition and proportion of carrier materials were determined (according to a singlefactor experiment) by the weight of mushroom slurry to be a soybean protein hydrolysate content of 10%, a gum Arabic content of 1% and a dextrin content of 15%.
4.3.2. Spray drying Reineccius (1988) suggested that for a successful microcapsulated product, even if the carrier materials were suitable, the yield of microcapsule product and the encapsulation effect might not be satisfactory if the optimum spray drying conditions were not used. The study indicated that the main factors in the spray drying were feed temperature, air inlet temperature, and air outlet temperature. Feed temperature affected the fluidity of the liquid, the difficulty of spray drying, the viscosity of the product and so on. We could increase the feed temperature to reduce the viscosity of liquid, which reduced the mean size of the droplets. However, the temperature should not be so high that it results in the volatilization of some volatile ingredients. We used a feed temperature of 50– 60 °C. Air inlet temperature has strong connection with the drying velocity and the ability to dry the microcapsule product, to achieve a granular structure with a suitable water content and the stability in the finished microcapsule product. The experiments showed that a suitable air inlet temperature was 130–140 °C. Beatus, Rziel, Rosenberg, and Kopelman (1985) suggested that when the air inlet temperature was low, the evaporation ability was not high enough to form the high density and intensive capsule membrane. At the same time, the product would have a high water content and have such a poor fluidity that it easily adhered to the wall; When the air inlet temperature was high, the excessive evaporation could easily cause a crack in the membrane as well as loss of flavoring ingredients through volatiliza-
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tion and the decomposition of heat-sensitive component at high temperature. This also causes off-flavors. Air outlet temperature had a significant effect on the product water content and the microencapsulated structure. Air outlet temperature must be controlled with the air inlet temperature. If the air outlet temperature was high, it will help to form an integrated and compact wall structure and thus improve the drying effect. Bhandari, Dumoulin, Richard, Nolean, and Lebert (1992) found that if the air outlet temperature were too high, the product would crack because of overheating. The product was ideal when the air outlet temperature was 70–80 °C.
4.3.3. Characteristics of the product The microencapsulated mushroom flavor product of our experiments was a globular micro granule that was light yellow in color and had a good dispersibility and solubility. Fifty particles selected at random were examined under a microscope. The particle size was about 40–90 lm, averaged at 65 lm; water content of the microcapsule was measured as 3.8%; the yield of microcapsules in our experiments was 92.1%.
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reason for these was that the flavor was not released unless heated or dissolved in water. 5. Conclusion (1) The preferred technology process to extract mushroom flavor was determined in our study. (2) The mushroom flavor could be strengthened by adding sunflower oil hydrolysate (mixed fatty acids) into the mushroom. The preferred conditions for enhancement were obtained from a single-factor experiment and an orthogonal experiment and selected as follows: amount of sunflower oil mixed fatty acids 0.3%, pH 6.5, heating time 40 min, temperature 35 °C. The flavor of the product was obviously strengthened under these conditions. (3) Spray drying was applied. The ratio of the carriers (calculated as the weight of fresh mushroom) was soybean hydrolyzing protein 10%, Arabic gum 1%, and dextrin 15%. Feed temperature, the air inlet and air outlet temperature during spray drying were 50–60, 130– 140 and 70–80 °C respectively. References
4.3.4. The results of the encapsulation effect as measured by organoleptic evaluation are stated in Table 4 The final result was examined by t-test, the value of t being 12.15. From the t-test tables we saw that t0:01 26 ¼ 2:4786, t0:005 20 ¼ 2:7787, so the value was far higher than the critical value (Table 4). In the evaluation results, the dry powder sample was remarkably different, the score for the dissolved sample being far higher than that of dry powder sample, which showed that the encapsulation effect was obvious to the panel lists. The
Table 4 Scores of encapsulation mushroom flavor product by organoleptic evaluation Judger
Dry powder ðS1 Þ
Dissolved in water ðS2 Þ
Difference
Difference squared
1 2 3 4 5 6 7 8 9 10 11 12 13 14
0 1 1 0 0 1 1 2 0 1 1 1 0 2
3 3 4 4 4 3 3 4 4 4 4 2 3 4
3 2 3 4 4 2 2 4 3 3 1 1 3 2
9 4 9 16 16 4 4 16 9 9 1 1 9 4
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