Sorne Factors Affecting Pectinase Production from Apple Pomace in ...

Biological Wastes 24 (1988) 147- 157

Sorne Factors Affecting Pectinase Production from Apple Pomace in Solid-State Cultures

R. A. Hours, C. E. Voget & R. J. Ertola Centro de Investigación y Desarrollo en Fermentaciones Industriales, Facultad de Ciencias Exactas UNLP, 47 y 115 (1900) La Plata, Argentina (Received 20 September 1987; accepted 12 October 1987)

ABSTRACT Somefactors affecting pectinase productionfrom apple pomace in solid-state culture (medium composition, inoculum size, temperature and type of apple pomace used) were investigated. To obtain high enzymatic activities, apple pomace requires the addition of an adequate leve! of organic nitrogen components like those present in peptone,yeast extrae! or corn-steep. Inoculum size is not critica! and optimum temperature for the fermenta/ion process is 30°C. In connection with applepomace quality, the sugar concentra/ion is the most importan! parameter which controls pectinase yields. By using Granny Smith pomace mixtures, containing 4% of sugars, production as high as 1300 U g - 1 was attained in 36 h of culture. Pectinase levels rangingfrom 1000 to 2000 U litre - 1 ofjuice for 1 to 3 h were necessary to achieve clarification in three different apple juices.

INTROD UCTION We recently reported the use of apple pomace for pectinases production in solid state cultures on a small scale (Hours et al., 1987). Yields as high as 1062 U g - 1 of apple pomace were attained at 48 h of culture with a strain of Aspergil/usfoetidus (NRRL 341, ATCC 16878) using a medium containing apple pomace, corn steep and salts. Under the culture conditions studied, the fungus produced an enzymatic pool containing polygalacturonases, pectin Biological Wastes 0269-7483/ 88/ $03·50 England. Printed in Great Britain

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147 1988 Elsevier Applied Science Publishers Ltd,

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R. A . Hours, C. E. Voget, R. J. Ertola

lyases and pectin esterases, which was considered adequate for practica! purposes in apple-juice industries. In this paper we report results related to the influence of medium composition, inoculum size, temperature and type of apple pomace used for pectinase production. In addition, the performance ofthe enzymatic pool in the clarification of apple juice is considered. METHODS The microorganism, inocula preparation, culture system, processing of samples and analytical techniques employed, including pectinase determination, were those described previously (Hours et al., 1988). In the present work, the percentage dry-weight loss was measured by the difference between the dry weights ofthe initial samples and those obtained during the process. The clarification tests were carried out with fresh apple juice prepared in our laboratory by pressing Red Delicious, Golden Delicious and Granny Smith apples with a hydraulic press with a cloth filter. The apples employed were commercial samples which were kept for 5 months in a cool chamber from the time of their harvest. After the pressing operation, the juice was heated to 80°C for 2 min to inactivate the native enzymes. For performing the test, 100 ml of juice containing a commercial amyloglucosidase (30 mg litre - 1 ), gelatine (50 mg litre- 1 ) and 1 ml of different dilutions of the pectinase crude extract obtained from the culture medium, were placed in 125 ml Erlenmeyer flasks and incubated at 40°C with gentle agitation. Samples were taken periodically, heated to 100°C for 3 min and centrifuged at 4000 g for 10 min. The transmittance at 660 nm was measured and iodine and alcohol tests were performed on the supernatant to check the development of the clarification process (Baumann, 1981; Meurens, 1979). The iodine test was carried out only in the case of Red Delicious apple juice, beca use, in the other two cases the juices did not contain starch detectable by this technique. Appropriate controls were done either without pectinase addition or with apple juice alone. Five types of apple pomace of different composition and origin were used as raw materials (Table 1). Samples lla and lila were taken after one pressing step, whereas samples I, llb and lllb were taken after three pressing steps and two washing treatments at the juice factory. In Argentina, the apple harvest takes place from February to April. So, during the April processing time, fruits are processed immediately after they are received, whereas in November, juice-plant processing is carried out using apples which ha ve been kept in cold-storage warehouses for 7 months.

Pectinase from apple pomace

149

TABLE 1 Composition• and Origin of Apple Pomace Samples

Type of apple pomace lb Humidity Crude fibre Crude fat Crude protein Soluble sugars Total pectic substances As hes pH Apples variety Apples processing time

85·2 2·63 1·50 0·45 3·7 1·80 0·25 4·1 RD April

!la

llb

83·1 89·0 1·96 2·13 1·58 1·38 0·38 0·44 9·1 2·1 1·20 1·20 0·29 0·21 4·1 4·1 RD RD November November

llla

lllb

80·6 3-97 0·80 0·35 9·1 1-30 0·27 3·3 GS April

90·5 2·81 1·20 0·39 1·4 0·86 0·24 J3 GS April

• On wet basis (g %). b This sample is the same as used in the previous work (Hours et al., 1988). RD, Red Delicious. GS, Granny Smith.

Samples Ila and lila are not representative of the normal pomace produced in the juice industries. The reason for their selection was to study a kind of raw material which eventually could be obtained in an apple-cider factory, where apples are pressed only once and the pomace is not washed. Consequently, as far as the Argentinian way of processing is concerned, samples I, Ilb and IIIb are typical of apple-juice processing plants and samples Ila and lila could be taken as belonging to a cider factory. Different by-products of the food industry were assayed as complex nitrogen sources. Their water and nitrogen contents, as percentage on wet basis, were, respectively, as follows: wheat bran (13·7, 2·7), wheat germ (13·0, 5·0), malt sprout (8·3, 4·3) rice bran (11·7, 2·6) and corn steep (1·0, 8·0). Other organic sources tested were yeast extract (Oxoid) and Bacto peptone (Difco). With most samples the initial pH ofmedia was not controlled because the values observed were appropriate for pectinase production. In the case of samples from Granny Smith apples, sorne processes were carried out with the pH adjusted to 4·1. All media were autoclaved at 121 °C, for 15 min. RESULTS AND DISCUSSION Influence of medium composition

Taking medium 1 with corn steep as a reference medium, six media containing other organic nitrogen sources were tested for pectinase

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TABLE 2 Influence of Medium Composition on Pectinase Production M edia

1a 2 3 4 5 6 7 8 9b

Nitrogen source (% wj w) NH4 N0 3

Organic

0·2 0·2 0·2 0·2 0·2 0·2 0·2 0·66 0·66

Corn steep Yeast extract Bacto peptone Malt sprout Wheat bran Wheat germ Rice bran Non e None

pH

2·0 1·6 1·1 7·0 6·9 3·7 4·0

Jnitial

Final

4·1 4·0 4·1 4·1 4·3 4·2 4·8 3-7 3·7

3·5 3·2 3·2 3·3 3·3 3-2 3·3 2·8 2·8

Pectinase activity (Ug - 1)

1 100 1250 900 250 230 135 130 130 50

Reference medium. Supplemented with the following salts (mg %): K 2 HP0 4 200; KCI 50; MgS0 4 . 7H 20 25; CaCI 2 .2H 20 20; Mo0 4 Na 2 . 2H 2 0 0·1; FeS0 4 .7H 2 0 0·05; ZnS0 4 .7H 2 0 0·04; CuS0 4 . 5H 2 0 0·04 (Mukherjee & Najumdar, 1974). All media contain apple pomace I with 0·05% ofNa 2 S0 4 . The nitrogen content ofmedia was 0·3%. Inoculum size; 10 5 spores g - 1 . Temperature, 30°C. Time of process, 48 h. a b

production, in a 48 h process using apple pomace I. The values obtained (Table 2) show similar yields of pectinase in the case of media containing corn steep, yeast extract or peptone. However, only 10% to 20% of this activity was attained with the other media. These results suggest that availability of organic nitrogen from these materials was insufficient, or higher levels of organic nitrogen fractions like those present in the former sources are required for good enzyme production. Poor results were also obtained with ammonium nitrate as the only nitrogen source, and a detrimental effect was observed when this medium was supplemented with micronutrients, which might possibly be dueto the presence of an element at inhibitory leve! (Meyrath & Volavsek, 1975). Pectinase yield was limited by corn steep when it was added in amounts lower than those used in the reference medium. Higher levels did not improve enzymatic production and the addition of (NH 4 hS0 4 (0·16%) or KH 2 P0 4 (0·1 %) to the reference medium had no effect on enzyme yield (data not shown). Effect of inoculum size It is known that inoculum size has a marked effect on enzyme yields in fungal cultures either in submerged or so lid state conditions (Meyrath & Suchanex, 1972).

151

Pectinase from apple pomace TABLE 3 Effect of Inoculum Size on Pectinase Production lnoculum size (spores per g of apple pomace)

103

Time (h)

o 12 24 36 48 72

104

o 12 24 36 48 72

105

o 12 24 36 48

106

72 o 12 24 36 48 72

pH

Glucosamine content (mgg - 1)

4·0 3·9 3·6 J3 3·2 3·7 4·0 3·9 3·3 3·2 3·2 3·6 4·0 3-8 N 3·3 N

0·02 0·02 0·03 0·30 0·55 0·98 0·02 0·02 0·06 0·33 0·67 0·95 0·02 0·03 0·09 0·36 0·75

J7

1·03 NO NO NO

4·0 3·8 3·2 3·1 3·2 N

ND ND

NO

Dry weight loss (mgg - 1)

Pectinase activity (Ug - t)

o o 11 ·0 16·0 43·0

45 920 1100 1150

o o

4·8 11·9 19·3 40·1

220 990 1120 1000

o o 6-4 12·8 21·0

300 990 1070

4JO

980

9·0 14·7 25·7 40·1

800 930 900 940

o o

ND, not determined. The processes were carried out with apple pomace type I in reference medium at 30°C.

In our case, inoculum size was varied 10 3 fold and although the rate of fermentation increased in the early stages of the process (Table 3), final maximum yields were quite similar and they were attained at the same time when inoculum size was changed from 10 3 to 10 5 spores g - 1 . Values of glucosamine indicate a quite good relationship between growth and pectinase production up to 36 h of culture, in which time 80% to 90% of the maximum activity was reached. At that time, dry weight loss which is related to the sugars metabolized for energy production, was 27% to 31% of the initial content of soluble sugars. After 36 h, growth is expected to be slower and the increase in the glucosamine and dry weight loss values may be mainly ascribed to the development of sporulation. Pectinase synthesis cea sed complete! y after about 48 h of culture. With the highest leve! of spores (10 6 sporesg - 1 ) about a 10% decrease in the maximum activity was

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observed. The fact that lower inoculum sizes do not affect enzyme production is very important because large production of spores becomes unnecessary. Influence of temperature The optimum temperature for pectinase production is 30°C (Table 4). At 25°C, fermentation rate was slower and enzyme yield was about 50% ofthat obtained in 48 h at 30°C. At 35°C growth took place normally, as is indicated by the glucosamine and dry weight loss values, but pectinase synthesis was markedly reduced. Elfect of apple pomace quality The results obtained with different types of apple pomace are shown in Table 5. Employing pomaces of high sugar content (types Ila and lila), the pectinase activity at 48 h was around 30% of the values attained with the pomace type l. In addition, reduced values of glucosamine and dry weight loss were also obtained, which indicate that metabolism and fungal growth were affected by these types of pomaces. Moreover, the activity did not increase with prolonged cultivation time. A similar effect was observed with pomaces of low sugar content (types Ilb and Illb), although in this case higher enzyme production occurred in the more acidic sample. When pomaces of the same type of apple were mixed in order to achieve a TABLE 4 Effect of Temperature on Pectinase Production Tempera tu re (OC)

25

30

35

Time (h)

pH

Glucosamine content (mgg - 1)

Dry weight loss (mgg - 1)

24 36 48 24 36 48 24 36 48

3-8 3-4 3-2 3·6 3·3 3-4 3·6 ND 3·0

0·039 0·106 0·29 0·07 0·32 0·70 0·16 ND 0·79

3·3 9-6 4·0 11-4 19-6 4·8 ND 24·1

Pectinase activity (Ug - 1) 7 207 615 217 980 1140 81 ND 93

ND, Not determined. Fermcntation process was done with apple pomace typc 1 in rcference medium. Inoculum; 104 spores g - 1.


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TABLE 5 Effect of Apple Pomace Type on Pectinase Production Apple pomace

Soluble sugars (mgg - t)

lnitial

Ila

41 91

4·0 4·1

Ilb lila

20 91

4·0 3·3

liJa• liib Il a (29 %) + Ilb (71 %) lila (34%) + Illb (66 %) lila (34%) + Illb (66 %)"

91 15

pH Final

Glucosamine content (mgg - t)

Dry weight loss (mg g - t)

Pectinase activity (Ug-t)

0·70 0·53 0·95b 0-49 0·55 1·27b 0·53 0·45

22·0 14-4 44·0b

4·1 3·3

H 3·0 3·5b 3·5 2·9 3·3 3·2 3·1

1190 360 320b 162 380 360b 312 290

40

4·1

3·3

41

3·3

3·1

0·75

22·0

1100

41

4·1

3·5

0·72

21 ·6

900

17·3 49·8b 19·5 20-4

750

• Initial pH adjusted by means of a NaOH solution. b Time of process; 72 h. The rest of the figures corrcspond to a 48 h process. Fermentations were done in reference medium, at 30°C. lnoculum; 104 spores g - 1 .

4% sugar concentration, the enzymatic production increased and, in the case ofthe Granny Smith pomace mixture (lila+ IIlb), the activity obtained was comparable with the reference one. Glucosamine data indicated that fungal growth was higher in this mixed sample. The effect of the initial culture pH was studied with pomace lila and the mixture above mentioned where pH was increased to 4·1. In both cases a slight decrease in pectinase activity was observed. These results indicate that, besides other factors, we have to consider the sugar concentration of the pomace as the most important factor which controls the yields of the fermentation under the culture conditions employed. So, we can assume that with sample IIlb, mycelial growth and enzymatic production were limited by the sugar con ten t. In samples Ila and lila, containing a sugar concentration as high as 9%, the results can be possibly ascribed toa negative effect on fungal metabolism ofunfavourable factors like high osmotic pressure of the medium. The time course of a process performed with a mixture of acidic pomaces is shown in Table 6. In this case, better pectinase activity and fermentation rate were obtained and the productivity increased 30% in relation to the pomace type I (reference medium, Table 3). Fungal biomass at 36 h can be

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estimated, as reported previously (Hours et al., 1988) 1as 5·2 and 6·1 mg of dried mycelium per gram of apple pomace in type I and in the mixture, respectively. So, pectinase yields are about 200 U per mg of dried mycelium. In order to compare our results with those obtained employing other substrates in solid-state cultures like wheat bran (Ghildyal et al., 1981), we evaluated the enzyme activity and calculated the units according to the authors mentioned. Thus, 1 unit of our work corresponds to 2·6 units in this paper. Although the raw material, strains and culture systems were different, our productivity per unit of area is 4·1 x 10 5 U h- 1 m- 2 , which is more than 10 times the values obtained in the above-mentioned paper. TABLE 6 Kinetics of Pectinase Production with a Mixture of Acidic Pomaces. Apple Pomace lila (34%) + Illb (66%) Time (h)

o 12 24 36 48

pH

Glucosamine content (mgg - t)

3·3 3·3 3·3 3·1 3·3

0·037 0·039 0·096 0-430 0·760

Pectinase activity (Ug-t)

o o 280 1280 1060

Fermentations were carried out with the reference medium, at 30°C. Inoculum; 104 spores g - 1 .

It is important to mention that at the time of maximum enzymatic production, the mycelial colonization of the media was still incomplete, in particular at the bottom section of the petri dish. In our culture system, spores are spread on the surface of media, so growth extends over it and progressively through the layer of substrate. Beca use of the pasty nature of the pomace, we ha veto consider that during the time course of the process the deepest fungal hyphae become seriously limited in oxygen nutrition. This produces an insufficient substrate utilization and, in addition, imposes a restriction on the amount of fermented material per unit of area. This effect is clearer if we consider that the layer of medium employed is only 0-4 cm thick compared with the typical values of2·5- 5·5 cm used in tray fermentors with wheat bran as substrate (Ghildyal et al., 1981; Lonsane et al., 1985). It is thus necessary to study the possibility of improving the culture system in relation to aeration efficiency; e.g. by modifying the rheology by mixing the apple pomace with inert materials in order to increase porosity (Aidoo et al., 1982).


155

Clarification of apple juice

The performance ofthe pectinase pool in the clarification of apple juices was tested with a crude extract obtained from a 36 h culture using the Granny Smith pomace mixture. Table 7 shows the production of the juice clarity with time, employing different levels of pectinase. Granny Smith juice was readily clarified in 1 h of treatment with 1000 U litre - 1 • At this time, abundant ftoc was observed and the alcohol test was negative. In the case of the Golden Delicious sample with the same level of enzyme addition, 2 h of incubation was necessary to obtain adequate clarity and a negative alcohol test. The situation was quite different with the Red Delicious juice because clarification was not completely achieved. In TABLE 7 Course of Transmittance at 660 nm with Time of Clarification of Three Apple Juices by A. f oetidus Pectinases

Time (min)

Pectinase added (U litre - 1 )

o 500 1000 2000 Control• Commercial sampleb Granny Smith apple juice, pH 3·3

o 500 1000 2 000 Control • Commercial sampleb Golden Delicious apple juice, pH 3·6

o 500 1000 2000 Control • Commercial sample b Red Delicious apple juice, pH 4·0

o

30

60

120

180

45 45 43 40 40 40

42

48

42

42

62 87 79

92 92 51 95

93 92 45 95

93 93 43 96

34 32 34 34 34 34

40 49 53 53 38 75

40 50 56 66 40 83

37 68 75 85 37 90

37 73 81 90 37 90

30 43 43

19 36 45 50

46

56

22 50 59 64 21 67

17 54 66 68 17 63

18 18 18 18 18 18

Juice alone. Bioconcentrada plus (Biocon. Ltd., Ireland); 1 000 U litre - 1 . All samples contained commercial amyloglucosidase (30 mg litre gelatine (50 mg litre - 1 ) .

240

21 65 66 18 73

a

b

1

)

and

156

R. A. Hours, C. E. Voget, R. J. Ertola

this case, using 2000 U litre - 1 for 3 h, the juice was still turbid even though the iodine test indicated that 2 h was sufficient for the saccharification ofthe starch present and the alcohol test was also negative.

CONCLUSIONS Apple pomace is an attractive raw material for pectinase production provided it is properly supplemented with an adequate organic nitrogen so urce. In order to scale up the process for commercial applications, further studies are required. In this respect, it is particularly important to test other strains, and also consider large-scale surface culture systems or bioreactor design if a bulk solid-state fermentation is to be applied.

ACKNOWLEDGEMENTS We wish to thank the Argentine Research Council (CONICET) and the Biotechnology Programme of the SECYT for the financia! support given, Mr Julio Figari for his technical assistance and 'Jugos del Sur' for supplying the apple pomace and commercial pectinase samples.

REFERENCES Aidoo, K . E., Hendry, R. & Wood, J. B. (1982). Solid substrate fermentations. In: Advances in applied microbiology. (Laskin, A. l. (Ed.)), Vol. 28, 201 - 37, Academic Press In c., New York, London. Bauman, J. W. (1981 ). Applications of enzymes in fruit juice technology. Enzymes Food Process (Ind. Univ. Co-op. Symp. 1980), 129-47. Ghildyal, N. P., Ramakrishna, S. V., Nirmala Devi, P., Lonsane, B. K. & Asthana, H . N . (1981). Large scale production of pectolytic enzymes by solid state fermentation . J. Food Sci. Technol. , 18 (6), 248- 51. Hours, R. A., Voget, C. E. & Ertola, R. J. (1988). Apple pomace as raw material for pectinases production in solid state culture. Biol. Wastes, 23, 221 - 8. Lonsane, B. K., Ghildyal, N . P., Budiatman, S. & Ramakrishna, S. V. (1985). Engineering aspects of solid-state fermentation. Enzyme Microb. Technol., 7, 258- 65. Meurens, M. (1979). Clarification of apple juices: In: Filtr. Beer other Beverage Ind. Symp., 227- 38. Meyrath, J. & Suchanex, G. (1972). Inoculation techniques: Effect dueto quality and quantity of inoculum. In: Methods in microbiology, Vol. 7B (Norris, J. R. and Ribbons, D . W. (Eds)), Academic Press, London, New York, 159- 210.


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Meyrath, J. & Volavsek, G. (1975). Production ofmicrobial enzymes. In: Enzymes in food processing (2nd edn) (Reed, G. (Ed.)), Academic Press Inc., London, 255- 300. Mukherjee, S. K. & Majumdar, S. K. (1971). Fermentative production ofpectinases by fungi: Screening of organisms and production ofthe enzymes by Aspergillus niger. J. Ferment. Technol., 49(9), 759- 70.