Die Nahrung 37 (1993) 6, 607-612

Microbial counts and invert sugars in juice extracts from stored tubers of Cyperus esculentus Linn. (earth almond)

L. 0. ADEBAJO

Department of Biological Sciences, Ogun State University, Ago-Iwoye, Nigeria

Summary

The microbial populations and the levels of invert sugars in juice extracts from healthy tubers of Cyperus esculentus L. stored at 10, 20, 30 and 40 "C were determined. Bacterial counts increased with time and attained the peak on the 25th and 35th d at 40 and 30 "C respectively. Similar trends were obtained for the yeasts, but the peak counts were recorded on the 20th and 35th d at 40 and 30 "C respectively. At 10 and 20 "C, the counts, both for bacteria and yeasts increased throughout the 40-d period. Invert sugar levels increased throughout the investigation period at all the tested temperatures. Highest levels were recorded at about 30 and 40 "C while the lowest levels were obtained at 10 "C. Of the seven bacteria and five yeasts tested, only Saccharomyces rouxii, S. cerevisiae, Schizo- saccharomyces sp., Pseudomonas chlororaphis, P. fluorescens and Bacillus subtilis brought about significant hydrolysis of sucrose in vitro. Results on the microbial populations and levels of invert sugars in raw juice extracts suggest that yeasts could possibly play a greater role than bacteria in the hydrolysis of sucrose in vivo.

Zusammenfassung

Keimzahlen und Invertzucker in Juice-Extrakten gelagerter Knollen von Cyperus esculenrus Linn. (Erdmandel)

Es werden die mikrobiellen Populationen und der Gehalt an Invertzucker in Juice-Extrakten aus gesunden Knollen von Cyperus esculentus L. bei der Lagerung bei 10,20,30 und 40 "C bestimmt. Die Bakterienzahlen nehmen mit der Lagerzeit zu und erreichen Maximalwerte am 25. bzw. 35. Tag bei 40 bzw. 30 "C. Ein ahnliches Verhalten wird fur die Hefen beobachtet, jedoch werden Maximalwerte am 20. bzw. 35. Tag bei 40 bzw. 35 "C erhalten. Bei 10 und 20 "C nehmen sowohl die Bakterien als auch die Hefe-Zahlen wahrend der 40tagigen Lagerung zu. Der Invertzuckergehalt erhoht sich wahrend der Lagerzeit bei allen gepruften Temperaturen. Die hochsten Gehalte werden bei etwa 30 bis 40 "C und die niedrigsten bei 10 "C gemessen. Von den gepriiften 7 Bakterien- und 5 Hefearten zeigen nur Saccharomyces rouxii, S. cerevisiae, Schizosaccharomyces sp., Pseudomonas chlororaphis, P. fluorescens und Bacillus subtilis eine signifikante Saccharosehydrolyse in vitro. Die Ergebnisse der mirkobiellen Populationen und Gehalte an Invertzucker in den rohen Juice-Extrakten deuten darauf hin, daI3 bei der Hydrolyse von Saccharose in vivo Hefen offenbar eine groI3ere Rolle spielen als Bakterien.

Introduction

Among the cheap and popular sweet convenience foods in West Africa is the rootstock snack or 'ofio'. It is essentially the washed and sun-dried tubers detached from the rhizomes of Cyperus esculentus or chufa. The plant, a nutsedge is a persistent perennial weed in many parts of the world [5, 6, 131. Prior to processing of the tubers, the water activity (a,) is usually favourable for the growth and activities of several microorganisms particularly

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1993 0027-769X/93/06 12-0519$5.00 + .25/0

608 Die Nahrung 37 (1993) 6

bacteria, the survival of which has been established in similar storage organs of some crops [14]. Such microbes gain entrance while the plant organs were still in the soil [4, 151 or they may invade through wounds at harvest time [lo, 121. During storage of the tubers, the sucrose content decreases and this leads to a corresponding fall in the juice quality. Such decrease in the sucrose content could be due to the metabolism of the inhabiting microbes and/or the biochemical activities of the tissue cells.

The present study was undertaken to determine the microbial populations and invert sugar levels in tubers of Cyperus esculentus with time of storage at 10, 20, 30 and 40 "C. The aim was to gain insight into the relevance of storage conditions on snack quality.

Materials and methods

Sample collection and preparation

Tubers of Cyperus esculentus were collected immediately after harvest from farms in and around Jos, Plateau State, Nigeria. A total of 25 samples were obtained and brought to the laboratory inside separate polythene bags. Each sample was divided into four portions and kept in separate troughs which were then stored inside incubators set at 10, 20, 30 and 40 "C.

Extrnction of juice

At the besinning of the experiment and at 5-day intervals, 300 tubers (approx. total wt. 118 g) were randomly selected from each sample stored at each of the four temperature settings. They were surface-sterilized by suspension in 1 YO sodium hypochlorite for 2 min and this was followed by rinsing in five changes of sterile distilled water before crushing (aseptically) in a mortar. 118 rnl of sterile distilled water was added and the juice was eventually extracted with a vegetable juicer fitted with a paper filter. The juicer was flushed with 70% alcohol and then with sterile water before and after each extraction to disinfect the device. Filters were replaced after four extractions. The amount of water added during extraction was taken into consideration when determining the concentration of the extracted juice in subsequent studies.

Determination qf microbial populations and levels of invert sugurs

Serial dilutions (10- to 10- 5 , of the raw juice extracted from each sample were aseptically prepared with sterile water and plated in triplicate by the pour plate technique. Nutrient agar and SABOURAUD agar were used for bacteria and yeasts respectively. Incubation was carried out at 25 and 37 "C for 2 to 3 d before colonies were enumerated. Subculturing was carried out to obtain pure cultures. Identification of bacteria was based on BERCEY [3, 71. Yeasts were also identified with the aid of appropriate manual [8].

The levels of invert (reducing) sugars in the extracted juice samples were determined by the 3, 5 dinitrosalicylic acid (DNSA) reagent [2]. This was achieved by adding 1 ml of DNSA reagent to each of 1 ml of juice extracts and the transmittance was determined at 540nm using an SP600 spectrophotometer. Transmittance was set at 100% with blank water-DNSA mixture. The DNSA reagent was prepared by adding. 1 .O g DNSA at 20 ml of 2 N NaOH and mixed with 20 g of potassium sodium tartrate dissolved in 100 ml of distilled water. Invert sugar concentrations were obtained from a standard curve constructed with increasing levels of glucose.

Release of invert sugars by isolates

This was determined to reveal the possible influence of each isolate on the juice quality during storage of the tubers. Freshly obtained juice extract was dispensed (5 ml/tube) separately in each of several test tubes. These were plugged with cotton wool and autoclaved at 1 1 5 "C for 30 min. The pH after

ADEBAJO: Juice extracts from stored tubers of earth almonds 609

autoclaving was 6.8. Isolates were singly inoculated using 0.2 ml cell suspension (2 x lo5 cells . m1-I) into each of the tubes which were then incubated at 30 "C. Control tubes were similarly prepared but the cell suspension was replaced with sterile water. At the beginning of the experiment and subsequently at 6-h intervals, the invert sugar levels were determined in triplicates by the DNSA reagent method.

Results and discussion

Microbial populations

Strains from six genera of bacteria and four yeasts were isolated (Fig. 1). The total counts obtained for each of the two microbial groups at harvest and when tubers were stored at 10, 20, 30 and 40 "C for a period of 40 d are presented in Fig. 2. On the average, the total count of either of the two groups in raw juice immediately after harvest was about 0.1 x lo4 m1-I. This count is comparatively lower than the average initial count recorded for bacteria in sugar beet tissues by MACDONALD et al. [9]. The results also show that population counts increased with time of storage at all the four temperature settings investigated (Fig. 2). For the bacteria, highest counts were distinctly recorded throughout the storage period at 40 "C while the same were obtained both at 30 and 40 "C for the yeasts. Despite the suitability of raw juice for microbial growth [4], relatively low maximum counts (<4.5 x lo4 rn1-I) were obtained in the current work thus suggesting a possible inhibitory influence of the tuber tissues on the growth of the inhabiting microbes.

Levels of invert sugars

The average level of invert sugars immediately after harvest was 0.6 mg . ml-I. This increased to about 5.5 mg . ml-' on the 40th d of storage at 30 and 40 "C (Fig. 3). Comparatively, the extent of inversion (hydrolysis) of the sucrose in the juice was low at 20 "C and even much lower at 10 "C. Inversion could have been catalysed by the enzyme invertase produced either by the inhabiting microbes or from tissues of the tubers or from both sources. According to MYRBACK [ 111, the exact optimum temperature for invertase activity is difficult to ascertain; however, it is expected to be around 30 and 40 "C which is close to the ambient temperatures (30 _+ 2 "C) in many parts of West Africa. Thus, a high level of invertase activity in the stored tubers is favoured. The increasing trends obtained both for microbial populations (Fig. 2) and levels of invert sugars (Fig. 3) suggest

1 C D h E F

Fig. 1 Occurrence of microbial genera in 112 cultures isolated from raw juice extracted from the tubers of,Cyperus esculentus. Bacteria: (A) Pseudomonas; (B) Alcaligenes; (C) Bacillus; (D) Chromobac- terium; (E) Flavobacterium; (F) Erwinia. Yeasts: (G) Saccharoniyces; (H) Schizosaccharomyces; (I) Candida; (J) Rhodotorula

I J

Die Nahrung 37 (1993) 6 610

B I 5,

A

Days stored D a y s stored

Fig. 2 Microbial populations in raw juice extracted from the tubers of Cyperus esculentus during storage of different temperatures. Each point is an average of 25 determinations

that tuber-inhabiting bacteria and yeasts could contribute to the inversion of sucrose during storage. Furthermore, the close similarity between the population growth curves at 30 and 40 "C (Fig. 2B) and the curves obtained for the levels of the invert sugars (Fig. 3) at the same temperatures is an indication that the yeasts are likely to be more important in the inversion process.

Release of invert sugars by isolates

Results obtained for the seven strains of bacteria and the five yeasts worked with are presented in Figs. 4 and 5, respectively. Apart from the Erwinia sp., Chromobncterium sp., Flavobacterium sp. and Rhodotorula sp. which showed very little or no ability to hydrolyse (in vitro) the sucrose in juice extract to reducing sugars, all the other cultures brought

O ' O 5 16 1 5 20 25 30 3k 40 Days stored

Fig. 3 Invert sugar levels in raw juice extracted from the tubers of Cyperus esculentus during storage at different temperatures. Each point is an average of25 determina- tions

ADEBAJO: Juice extracts from stored tubers of earth almonds 611

0 12 24 36 48 60 72 Incubation t ime ( hours

Fig. 4 Relase of invert sugars in autoclaved juice by the bacterial isolates with time at 30 "C. Each point is an average of three replicate determinations. Alcaligenes sp. ( 1 ) ; Bacillus subtilis ( 2 ) ; Chromobacterdum sp. (3); Erwinia sp. (4); Flavobacterium sp. ( 5 ) ; Pseudomonas chlororaphis ( 6 ) ; P.f(uorescens (7 )

about the hydrolysis, though at varying extents. The fairly static levels of invert sugars recorded with time for most of the test cultures could be due to the conversion of the sugars to other metabolites. A negative feed-back effect resulting from the attendant fall in a, as a result of the increasing reducing sugar level in the medium is another possibility. The partial or total absence of invertase activities recorded for the four organisms earlier mentioned could be characteristic of the strains worked with or as a result of unfavourable environmental condition(s) such as adverse pH level. Indeed three invertase types have been identified based upon the pH preference; acid, alkaline and neutral [l].

Although the current results may not represent precisely the microbial hydrolysis of sucrose in vivo, however, they are of economic and practical significance. Tubers should be processed immediately after harvest to maintain high juice and taste quality. When this

6 1

0 12 2L 36 48 60 72 Incubation t ime (hours )

Fig. 5 Release of invert sugars in autoclaved juice by the yeast isolates with time at 30 "C. Each point is an average of three replicate determinations. Candida sp. (1); Saccharomyces cerevisiae (2); S. rouxdi (3); Schizosaccharomyces sp. (4); Rhodotorula sp. ( 5 )

612 Die Nahrung 37 (1993) 6

is not possible, low temperature storage would retard the rate of sucrose inversion and thus sustain the desirable sweet taste. The latter probably explains the consumers’ preference for the rootstock snack prepared from tubers cultivated and stored in the cool region of Plateau State, Nigeria, where the ambient temperature is about 20 “C.

References

[ I ] ALEXANDER, A. G., Sugarcane Physiology. Elsevier Scientific Publ. Comp., Amsterdam 1973. [2] BERNFELD, P. C., Adv. Enzymol. 12 (1951) 379-428. [3] BERGEY’S Manual of Determinative Bacteriology. Ed. by R. E. BUCHANAN and N . E. GIBBONS.

8th ed. Williams and Wilkins Co., Baltimore 1974. [4] FRAZIER, W. C., and D. C. WESTHOFF, Food Microbiology. 3rd ed. Tata McGraw-Hill Publishing

Company Ltd., New Delhi 1978. [ 5 ] GHAFAR, Z., and A. K. WATSON, Weed Science 31 (1983) 572-575. [6] GLAZE, N. C., C. C. DOWLER, A. W. JOHNSON and D. R. SUMMER, Weed Science 32 (1984) 762 - 767. [7] BERGEY’S Manual of Systematic Bacteriology, Vol. 1. Ed. by N. R. KRIEG and J. G. HOLT. Williams

[S] LODDER, J., The Yeasts. A Taxonomic Study. 2nd ed. North-Holland Publishing Company,

[9] MACDONALD, I. R., D. M. WEBLEY and J . S. D. BACON, Biochem. J. 89 (1963) 204.

and Wilkins Co., Baltimore 1984.

Amsterdam, London 1970.

[lo] MCDONALD, D., Rev. Appl. Mycol. 48 (1969) 465-474. [ 111 MYRBACK, K., in: The Yeast. Vol. 2. Ed. by A. H. ROSE and J . S. HARRISON. Academic Press,

[12] OKOLI, C. A. N., and I. D. ERINLE, J. Stored Prod. Res. 26 (1990) 77-79. [13] REDDY, K. N., and L. E. BENDIXEN, Weed Science 37 (1989) 147-151. [I41 TERVET, I. W., and J. P. HOLLIS, Phytopathol. 38 (1948) 960-967. [15] VINCELLI, P. C.. and J. W. LORBEER, Mycopathologia 111 (1990) 67-72.

Dr. L. 0. ADEBAIO, Department of Biological Sciences, Ogun State University, P.M.B. 2002, Ago-lwoye, Nigeria

Received November 20, 1992 Revised manuscript received August 4, 1993

London 1970.