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Materials and methods


Substrate

Ripe and unripe Cavendish bananas in slurry form, used as substrate for fermentation, were prepared as follows: A known weight of bananas (whole fruit including peels) was chopped into small pieces and pureed with distilled water in a blender to make a slurry with a banana-to-water ratio of 1:3. The following supplements were added: (NH4)2SO4, 4 g/litre KH2PO4, 2 g/litre; MgSO4, 1 g/litre; and calcium pantothenate, 4.5 mg/litre. The pH of the slurry was between 4.5 and 4.9.

The Organisms

Four strains of Aspergillus niger (UPCC 3701, 3026, 3450, and 3809), two strains of Aspergillus foetidus (UPCC 3702 and 3448), and mixed cultures of Endomycopsis fibuligera (UPCC 2407) with Candida utilis (UPCC 2074) and of A. foetidus (UPCC 3448) with A. niger (UPCC 3809) were used as test organisms. Fungi were maintained on Ozapek Dox agar slants and yeasts on yeast malt agar slants at 29 to 30C. These organisms were obtained from the culture collection of the University of the Philippines Natural Research Center.

Preparation of the Inoculum

Primary Inoculum

A.niger and A. foetidus were cultivated on Ozapek Dox agar slants, while E. fibuligera and C. utilis were cultivated on yeast malt agar slants. After three or four days of incubation at 29 to 30 C, spore suspensions of the organisms were transferred aseptically to bottles containing the supplemented substrate solidified with agar. This was done to acclimatize the organisms to the substrate. The inoculated bottles were incubated at 29 to 30C for three or four days. The spore suspension prepared from these bottles was used as the primary inoculum.

Secondary Inoculum

The secondary inoculum was prepared by adding 5 ml of the primary inoculum to 10 ml of sterile banana slurry in a 500-ml Erlenmeyer flask, which was then shaken for 24 hours. After that, 60 ml of sterile medium was added, and shaking was continued for another 24 hours. Then 80 ml of fresh, sterile substrate was added, to make up a total volume of 150 ml, and the flask was again shaken for 24 hours The resulting culture was used as inoculum.

For succeeding fermentation runs, to minimize the time lag between runs, the inoculum was prepared by inoculating 150 ml of substrate with 10 to 15 ml of primary inoculum and shaking it for 24 hours. For fermentations on a larger scale, the inoculum was scaled up correspondingly.

Batch Culture Fermentation

Fermentation was carried out for 24 hours in a 5-litre reactor vessel (Marubishi Ltd., Japan) with a working volume of 2.5 litres. The pH was maintained at between 4.0 and 5.0 by automatic addition of NH4OH. Air flow rate was controlled from 0.5 to 5.0 litres/min, and agitation speed was regulated from 200 to 600 rpm to maintain the dissolved oxygen concentration above 1 ppm. Changes in pH, sugar concentration, and biomass concentration were noted at regular intervals (every four hours). Foam was controlled by the automatic addition of an antifoaming agent, while temperature was maintained at approximately 30C.

Fermentation runs were carried out on a larger scale in a 14-litre Microferm fermentor (New Brunswick Sci. Co., USA) with a working volume of 7 litres. It has automatic pH, dissolved oxygen, temperature, and foam control. Maximum air flow rate values of 16 litres/min and agitation speeds up to 1,000 rpm can be attained. A 10 per cent v/v inoculum size was used.

Analytical Procedure

A 10 ml portion of every sample taken was centrifuged. The supernatant was collected and analysed for sugar content by means of the Somogyi-Nelson method of reducing. sugar determination (35; 36). The volume of residue was noted, and this was washed three times with 0.9 per cent saline solution and dried to constant weight at 60 to 80C in a vacuum oven in aluminium foil boxes previously dried to constant weight. The dried samples were analysed for their crude protein content by the micro-Kjeldahl method (37).

Results and Discussion

The main pulp of the Cavendish banana contains a considerable amount of carbohydrates, mostly starch and reducing sugars, but is low in protein. The peels have a crude fibre content of 2.08 per cent (unripe) and 1.93 per cent (ripe). The extremely large reserves of polysaccharides make the banana rejects a potential source of SCP.

Two methods were proposed for using the banana rejects (fig. 2). The first involves treating the bananas with dilute acid in an autoclave, which results in the breakdown of starch and cellulosic materials into simple sugars. The hydrolysate obtained is used as substrate for SCP production, giving rise to a product consisting wholly of fungal mycelia or yeast cells. The second method is the direct enzymatic fermentation by selected fungi and yeasts of slurry prepared from the bananas. The product obtained in this method consists of yeast cells or fungal mycelia plus unhydrolysed banana residues. All of the investigations carried out made use of the whole banana fruit (pulp and peel), as it would not be economical if only the pulp or the peels were used.

Acid Hydrolysis

Studies on the acid hydrolysis of bananas were conducted. The effects of several factors, namely, acid concentration, banana-to-water ratio, and time (duration) of hydrolysis, on sugar yield were studied. Hydrolysis was carried out at 121C because results of studies done in our laboratory on the acid hydrolysis of rice straw 138) as well as those reported in the literature (24) showed that the yield of sugar at this temperature was 30 per cent higher than the yield at 100C at all the acid concentrations used

FIG. 2. Use of Banana Wastes for the Production of Single-Cell Protein (SCP) by Microbial Processes

Results show that maximum reducing-sugar yield (as glucose) from dried, unripe bananas was obtained when banana diluted with water to a 1:2 ratio was treated with 4 per cent H2SO4 for 30 minutes, or with 2 per cent H2SO4 for 1 hour at 121C. Dried, ripe bananas had lower sugar yields when subjected to the same conditions of hydrolysis. This is because starch is converted to simpler sugars as the banana ripens, and acid hydrolysis of the ripe banana results in further degradation of these sugars to produce furfural and other degradation products that could be inhibitory to microbial growth. Hence, ripe bananas do not need to be hydrolysed in order to prevent the production of toxic by-products. Much of the reducing sugars present in ripe bananas can be directly used by micro-organisms for SCP production.

TABLE 2. Fermentation Data on Some Yeasts and Fungi Grown on Banana Slurry by the Batch Culture Method

 

Crude Protein Content (N x 6.25) of Product

(%)

Crude Protein Yield (g/ml)

Conversion of Substrafe to Crude Protein

(%)

Aspergillus niger (UPCC 3450)

ripe

19.25

0.0048

17.20

 

unripe

28.02

0.0065

15.82

A. niger (UPCC 3026)

ripe

30.67

0.0076

32.90

 

unripe

19.88

0.0046

15.23

A. niger (UPCC 3701)

ripea

27.51

0.0085

40.43

 

unripe

22.25

0.0054

21.01

A. niger (UPCC 3809)

ripea

25.31

0.0065

26.29

 

unripe

23.85

0.0066

19.41

A. foetidus (UPCC 3448)

ripe

27.83

0.0083

45.11

 

unripe

20.88

0.0055

14.82

A. foetidus (UPCC 3702)

ripe

26.58

0.0063

31.50

 

unripe

21.75

0 0050

13.89

Endomycopsis fibuligera

       

(UPCC 2407) and Candida

ripe

30.08

0.0070

24.05

utilis (UPCC 2074)

unripea

30.26

0.0076

16.02

A. foetidus (UPCC 3448)

ripea

41.30

0.0082

57.85

and A. niger (UPCC 3809)

unripea

32.09

0.0080

31.36

a. Average values of several fermentation runs.

Studies on acid hydrolysis were later abandoned because this process does not appear to be economically feasible. Considering the amount of acid, heat, water, and time required for the process, this method is an expensive step in the production of SCP. Thus, the emphasis in the present research project is confined to direct enzymatic fermentation by micro-organisms.

 

Direct enzymatic fermentation


One phase of this research that is being investigated is concurrent enzymatic starch hydrolysis and yeast cell multiplication through the use of a mixed culture. This process would be cheaper than starch hydrolysis by acid followed by yeast propagation and is accomplished by inoculating the banana slurry with equal volumes of Endomycopsis fibuligera, a mycelial yeast that produces an amylase capable of breaking down starch into glucose, and Candida utilis, a known food yeast that cannot use starch directly but can use glucose.

Growth of these organisms on unripe banana substrate increased the crude protein content of the final product (yeast cells and unhydrolysed banana residues) to an average value of 30.26 per cent. On ripe bananas, average final crude protein content was 30.08 per cent. The fermentation data are shown in table 2. The relevant equations are:

protein yield =[% crude protein]/100 x product yield (final dry weight)

conversion efficiency = [protein yield] / initial dry weight x 100

During fermentation, considerable weight loss is observed with unripe banana substrate, while there is a slight decrease in dry weight when ripe bananas are used.

Dry weight measurements, however, cannot be used to follow the growth of the organisms since the samples are a mixture of microbial cells and unhydrolysed banana residues that cannot be separated. The behaviour of dry weight measurements can be interpreted in terms of a balance between microbial growth, which tends to increase dry weight, and hydrolysis of the banana slurry, which tends to decrease dry weight of the product, The final product is lower in dry weight because of incomplete conversion to microbial cells of the hydrolysed banana substrate. Part of it is metabolized to other products such as CO2 and residual sugars or is converted to energy (as ATP), which is required for hydrolysis.

On both ripe and unripe bananas, reducing-sugar concentration in the supernatant remains very low (less than 0.003 g/ml) throughout the fermentation period (fig. 3). This is because C. utilis immediately utilizes the sugars released by E. fibuligera from the banana substrate.

Fig, 3. Fermentation Patterns of a Mixed Culture of Endomycopsis fibuligera (UPCC 2047) and Candida utilis (UPCC 2074) on Ripe and Unripe Banana Slurry. A: On ripe bananas. B: On unripe bananas.

^ = reducing-sugar concentration (as glucose, g/ml).
= dry weight.

Two other organisms used mixed culture are A. niger 3809 and A. foetidus 3448. A. niger is known to produce large amounts of amylases and also cellulases; A. foetidus produces only small amounts of amylases; and both can use glucose equally well. In contrast to the mixed culture of two yeasts, reducing sugar is found to accumulate in the medium. When unripe bananas are used, rapid enzymatic hydrolysis occurs during the initial growth phase (fig. 4B). Sugar concentration increases rapidly during the first 12 hours (0.0002 to 0.0167 g/ml, or an 80-fold increase), which is accompanied by a sharp decline in dry weight. Dry weight then increases with time with a corresponding decrease in sugar concentration.

In the case of ripe bananas, despite the high concentration of sugar initially present (0.0242 9/ml), slight hydrolysis still occurs during the first eight hours, causing the sugar concentration to increase to 0.0292 g/ml with a corresponding decrease in dry weight (fig. 4A). This may mean that the enzymes are not inhibited at this sugar level. Rapid utilization of the sugar follows, accompanied by an increase in dry weight. Accumulation of sugar in the medium is not surprising, as both organisms are amylase-producing.

FIG. 4. Fermentation Patterns of a Mixed Culture of Aspergillus niger (UPCC 3809) and A. foetidus (UPCC 3448) on Ripe and Unripe Banana Slurry. A: On ripe bananas.

B: On unripe bananas.

^ = reducing-sugar concentration (as glucose, g/ml).
= dry weight.

The second phase under investigation is the direct fermentation of bananas into microbial protein by one organism alone. Different isolates of A. niger and A. foetidus were used in this study. Results are shown in table 2.

When A. niger 3809 is used alone, final dry weight is lower for both ripe and unripe bananas, although the same changes in dry weight during fermentation are observed as in the mixed culture of the two fungi (fig. 5A and B). The same general behaviour of reducing sugar in the medium is also observed, With ripe bananas, two peaks are observed, one at 4 and the other at 12 hours. However, the increase in sugar concentration is small (0.0355 to 0.0412 and 0.0380 g/ml). In the case of unripe bananas, the peak is reached 8 hours after inoculation (0.0052 to 0.0165 g/ml, representing a threefold increase in sugar concentration), with a smaller peak at 16 hours (0.0093 g/ml).

When A. foetidus 3448 is used alone on ripe bananas, a very slight peak in sugar concentration appears after 4 hours (0.0178 to 0.0201 g/ml) accompanied by a slight decrease in dry weight (fig. 5C and D). In the case of unripe bananas, final dry weight is lower, with the dry weight changing gradually during the fermentation, as shown by a smooth, rounded curve, and the peak in sugar concentration occurring only after 20 hours (0.0053 to 0.0101 g/mg, or a twofold increase), which probably indicates slow hydrolysis of the banana. This confirms an earlier statement that the amylase of A. foetidus 3448 is weaker than that of A. niger 3809.

FIG. 5. Fermentation Patterns of Single-Organism Cultures of Aspergillus niger (UPCC 3809) and A. foetidus (UPCC 3448 and 3702) on Ripe and Unripe

Banana Slurry.

A: A. niger 3809; ripe.
B: A. niger 3809; unripe.
C: A. foetidus 3448; ripe.
D: A. foetidus 3448; unripe.
E: A. foetidus 3702; ripe
F: A. foetidus 3702; unripe
^ = reducing-sugar concentration (as glucose, g/ml).
= dry weight.

FIG. 6. Fermentation Patterns of Single-Organism Cultures of Aspergillus niger (UPCC 3701, 3450, 3026) on Ripe and Unripe Banana Slurry.

A: A. niger 3701; ripe.
B: A. niger 3701; unripe.
C: A. niger 3450; ripe.
D: A. niger 3450; unripe.
E: A. niger 3026; ripe.
F: A. niger 3026; unripe.
^ = reducing-sugar concentration (as glucose, g/ml).
= dry weight.

We can conclude that the use of A. foetidus 3448 and A. niger 3809 in combination is better than using either one alone, because the crude protein content of the final product and the efficiency of conversion of substrate to protein is higher when the mixed culture is used, whether with ripe or unripe bananas.

A. foetidus 3702 behaves in a similar manner to A. foetidus 3448 on both ripe and unripe bananas (fig. 5E and F). There is very slight hydrolysis of the substrate, indicated by the absence of sharp peaks in reducing-sugar concentration in the medium. A. foetidus 3448 may even be slightly better than A. foetidus 3702 because more sugar accumulates in the medium when the former is used. It has also been observed that the crude protein content of the products and conversion efficiency of the two organisms are similar,

When the three remaining strains of A. niger (3701, 3450, and 3026) are compared, final dry weight is higher than the initial weight on ripe bananas for A. niger 3701, lower for A. niger 3450, and approximately the same for A. niger 3026 (fig. 6). A. niger 3026 produces the highest peak in reducingsugar concentration after 8 hours (0.0465 to 0.2789 g/ml, for a sixfold increase), while the dry weight hardly changes. For A. niger 3701, a small peak occurs at 8 hours, with the dry weight surprisingly reaching a peak at this point also. This may be attributed to sampling error With A. niger 3450, a rapid decline in sugar concentration prior to the occurrence of a peak that is lower than the initial value is accompanied by a decrease in dry weight.

Two peaks in sugar concentration are always observed, a large peak during the initial growth phase and a smaller peak after most of the sugar has been used up. This may be attributed to increased activity of the starch-hydrolysing enzymes when the sugar concentration drops to a low level or to decreased growth rate of an organism that has probably reached the stationary phase while enzyme activity remains the same, leading to accumulation of the released sugar in the medium. This is not observed with A. niger 3809.

On unripe bananas, the final dry weight is lower for all three organisms. No appreciable peaks in sugar concentration are observed, indicating that the hydrolysis taking place is just enough to support microbial growth. A. niger 3809 may have stronger amylase activity on this substrate, since more sugar is released Into the medium (0.0052 to 0.165 g/ml).

When the crude protein content of the final product and conversion efficiency are compared, growth on ripe bananas generally produces higher values than on unripe bananas. A. niger 3701 has the highest average crude protein content on ripe bananas (27.51 per cent). The value for A. niger 3026 was taken from one fermentation run only and so cannot be interpreted as the highest. A. niger 3701 also has the highest conversion efficiency. On unripe bananas, A. niger 3450 has the highest protein content, but A. niger 3701 has the highest conversion efficiency.

For all the fermentation runs performed, the final product is almost always lower in dry weight than the starting material when unripe banana slurry is used as substrate. In some cases, reduction in weight of more than 50 per cent is observed. In the case of ripe bananas, final dry weight may be higher or lower. In contrast to the mixed culture of yeasts, accumulation of reducing sugar in the supernatant is always observed sometime during the fermentation when fungi are used. This drops to low levels as the fermentation continues.

A mixed culture of A. foetidus 3448 with A. niger 3809 appears to be the best on ripe and unripe bananas, followed by a single culture of A. niger 3701 and a mixed culture of E. fibuligera 2047 with C. utilis 2074. However, definite conclusions cannot be drawn until a more direct measurement of starch utilization has been carried out. A method of starch analysis is being studied, and we hope analyses can be completed in the near future. An indirect method of following microbial growth - analysis of the protein content during fermentation - is also being studied. Relative nutritive values of the different types of microbial protein produced cannot be compared at present because analyses of the amino acid contents and toxicological tests have yet to be carried out.

Results of this research so far have demonstrated the possibility of growing fungi and yeasts on banana waste with relatively high crude protein content. However, it is not comparable to that of current protein sources such as plant proteins and commercial animal feeds (e.g., soy meal, 45 to 50 per cent; fish meal 60 to 65 per cent). It has also been shown that no preliminary chemical treatment is necessary as long as the appropriate organisms are used. Further studies will be conducted in an attempt to increase the protein content and conversion efficiency.

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