European J. Appl. Microbiol. Biotechnol. 10, 253--258 (1980)

European A r ~ l i , , ~ r i Journal of h"k~l;~l I~11

Microbiology and Biotechnology �9 by Springer-Verlag �9 1980

Effect of Lysozyme and Sodium EDTA on Shrimp Microflora

Ramesh Chander and Norman F. Lewis

Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Bombay 400 085, India

Summary. The influence of lysozyme and salts on the growth of the micro- flora of shrimp was investigated�9 It was found that lysozyme at concentrations up to 150 #g/ml could retard microbial growth in nutrient broth at 28~ Growth of shrimp microflora was not affected much at low concentrations (0.05% and 0.1%) of EDTA but was totally inhibited in the presence of 0.5% Na2EDTA. No growth was discernible using concentrations of 50 gg/mi lyso- zyme and 0.02% Na2EDTA, either in nutrient broth or in 2% shrimp homo- genate.

Introduction

Lysozyme, because of its bactericidal properties, has been suggested for use as a preser- vative in the food industry. It has the essential advantage of being found in almost all human and animal cells and does not have harmful effects. Eisai Co. Ltd. (1966) pat- ented their process of preservation of meat products using Iysozyme, white Igarashi and Zama (1972) patented their method of using lysozyme for the preservation of edible sea-foods. Hen egg-white lysozyme is presently the most important commercially available lysozyme.

The present investigations were undertaken to ascertain the suitability of lysozyme for use in shrimp preservation. Spoilage of shrimp during storage has mainly been at- tributed to the growth and proliferation of microorganisms. Love and Thompson (1965) showed that the microbial flora of fresh iced shrimp is primarily made up of Gram-negative bacteria belonging mainly to the genus Pseudornonas, Acbromobacter, and Flavobacterium and that predominant microorganisms shift during storage. Vander- zant et al. (1970) reported predominance of coryneforms Pseudomonas, Maraxella, and Micrococcus species in Gulf shrimp.

O171-1741/80/0010/0253/~ 01.20

254 R. Chander and N.F. Lewis

Materials and Methods

Preparation ofFish Homogenate

Fresh shrimp (Penaeus indicus) purchased in the local market at Bombay were brought

to the laboratory on ice. After peeling and deveining, they were thoroughly washed in running tap water. Ten grams quantities of shrimp were added to 90 ml sterile distilled

water and homogenised for 2 min using a Sorvall Omnimixer.

Preparation of "Active Shrimp Culture"

Five ml of 10% shrimp homogenate was inoculated into 45 ml nutrient broth in 250 ml conical flasks. The flasks were then incubated at room temperature (28~ on a rotary shaker for 14-16 h to ensure that the cells would be in the logarithmic phase of growth, This 16 h biomass designated as "active shrimp culture" which was used

for further investigations, had a cfu number of 1-2 x 106 m1-1.

Lysozyme Treatment

Lysozyme at appropriate concentrations was added aseptically to 30 ml sterile nutrient broth kept in 250 ml conical flasks fi t ted with a side arm. These flasks were inoculated with 1% inoculum of active culture and growth was measured at regular t ime intervals,

as described below.

Salt Treatment

Various salts including sodium chloride and sodium EDTA were added individually to the nutrient broth in appropriate concentrations. Of salt containing nutrient broth 30 ml quantities were distributed in conical flasks with side arms and sterilized. To these, 1% inoculum of active shrimp culture was added and growth was measured at

regular intervals.

Measurement of Growth of Active Culture

The growth of active shrimp culture was assessed by measuring OD at 600 nm using a Spectronic 20. For rapid measurement, the side arms of flasks were directly put into

the cuvette holder. Similar uninoculated flasks served as blanks.

Results and Discussion

~ffect of Lysozyme The active shrimp culture was inoculated into nutrient broth containing 0-150/ag/ml lysozyme and OD measured at regular intervals. The results, shown in Fig. 1, demon- strate that a normal growth pattern was obtained in control falsks (no iysozyme) but on addition of lysozyme, the OD instantly increased from 0.02 to 0.2 and within 2 h dropped down to the original level, apparently giving false readings. A similar rise in OD of suspensions of Lancifield D streptococci in distilled water on lysozyme addition was reported by Metcalf and Deibel (1973). However, these workers noted that the change was stable for several days. I t was observed that small amounts of sodium chloride in nutrient broth prevented the initial rise in absorbance, hence the growth of

Effect of Lysozyme and Sodium EDTA on Shrimp Microflora 255

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Fig. 1. Effect of lysozyme on the growth of shrimp microflora in nutrient broth. Lysozyme (0- 150 #g/ml) was added aseptically to 30 ml sterile nutrient broth. This was then inoculated with 1% inoculum of 'active culture' and growth was observed by measuring OD at 600 nm

Fig. 2, Effect of lysozyme on the growth of shrimp microflora in nutrient broth containing 0.05% NaC1. Lysozyme (0 150/lg/mi) was added aseptically to 30 ml sterile nutrient broth which con- tained 0.05% NaCI. This was inoculated with 1% inoculum of 'active culture' and OD was measured at 600 nm

the active culture was determined in nutrient broth containing 0.05% NaCl and various

lysozyme concentrations. These results are graphically represented in Fig. 2. Lysozyme

addition resulted only in slight retardation of growth as compared with the control

flasks. Similar observations were made with Streptococcus faecium strains which dis-

played normal growth in nutrient broths containing 200/~g/ml lysozyme (Metcalf and

Deibel 1972). However, two strains of Pediococcus bomari and one strain of Micro- coccus lysodeikticus did not grow in TYE containing 100 ;tg lysoyzme per ml (Metcalf and Deibel 1972).

Effect of Sodium Cbloride

As mentioned earlier, the addition of NaC1 was found essential prior to lysozyme in-

corporation in the medium. The influence of various NaC1 concentrations on the

growth of active shrimp culture was determined therefore. NaC1 from 0 to 3% was

incorporated in nutrient broth and the increase in OD of the active shrimp culture

observed at various intervals. As can be seen in Fig. 3, NaC1 enhanced the growth

rate of the culture. Hence, only the minimum amount of NaCI (0.5%) was used in further experiments.

Effect of Disodium-ED TA

The major fish spoilage microflora are Gram negative (Love and Thompson 1965;

Vanderzant et al. 1970; Alur et al. 1971). Because of their cell envelope, lysozyme

256 R. Chanderand N.F. Lewis

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Fig. 3. Effect of sodium chloride on the growth of shrimp microflora. Sodium chloride from 0- 3% (W/V) was added to nutrient broth and sterilized. 1% inoculum of 'active culture' was added and OD600 nm measured at various intervals

Fig. 4. Effect of disodium-EDTA on the growth of shrimp microflora. Disodium-EDTA was added from 0-0.5% concentration into nutrient broth and sterilized, This was then inoculated with 1% inoculum of 'active culture' and OD600 nm measured at various time intervals

cannot penetrate easily into Gram negative cells; hence, some physical or chemical

treatments are used to sensitize Gram negative bacteria to lysozyme.

It is well documented that in buffer systems, EDTA can sensitize some bacteria to lysozyme action (Repaske 1958). However, it is not reported whether EDTA can sen-

sitize microorganisms to lysozyme action in a rich nutrient medium. Hence, the response of active shrimp microflora to disodium EDTA alone or in com-

bination with lysozyme was examined. For this purpose, active culture was added to

nutrient broth containing 0% to 0.5% disodium EDTA. The results are shown in Fig.

4. As seen in Fig. 4, 0.5% Na2EDTA could totally control microbial growth, whereas lower concentrations (0.1% and 0.05%) could only retard growth. The flasks containing

0.02% Na2EDTA exhibited a growth pattern similar to that of control flasks (Na2EDTA).

EDTA has also been reported by Levin (1967) to inhibit psychrophilic bacteria in

nutrient broth.

Effect of Lysozyme and EDTA

Concerning the toxicity of EDTA, there seems to be no objection to the daily use of the calcium salt of EDTA at levels not exceeding 1.25 mg/kg body weight (FAO/WHO 1967). General use of disodium EDTA as a food additive is not recommended unless used soley for accurately complexing calcium ions, and no excess of, Na2EDTA must remain (FAO/WHO 1967). Thus it is important to know whether the lowest concen- tration of EDTA could show synergistic effect with lysozyme in preventing the growth of shrimp microflora.

Hence, growth was measured in the presence of 0.02% Na2EDTA (which alone could not prevent the growth) and 50 I~g/ml of lysozyme. As shown in Fig. 5, no growth of

Effect of Lysozyme and Sodium EDTA on Shrimp Microflora 257

Fig. 5. Effect of lysozyme and 0.02% Na2EDTA on the growth of shrimp microflora. Lysozyme in varying concentrations (0-150 tag/ml) was added aseptically to 30 ml sterile nutrient broth contain- ing 0.02% Na2EDTA. OD at 600 nm was then measured at different intervals of time after inoculating 'active culture' at 1% level of inoculum

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the active shrimp microflora was observed for 48 h in the presence of this combination, while the untreated culture displayed normal growth pattern. Furthermore, the OD of the treated flasks remained constant for many days - a finding not shown in Fig. 5.

Effect of Lysozyme and Na2EDTA on the Growth of Sbrimp Culture

As the combination t reatment (50/~g/ml and 0.02% Na2EDTA) consistently prevented growth of active shrimp culture in nutrient broth, the nutrient broth was replaced by sterile 2% shrimp homogenate with 0.5% NaC1, to study whether the organic matter of shrimp can modify the growth. Even in 2% shrimp homogenate, the active culture could not grow in the presence of 50/.tg/ml of lysozyme and 0.02% Na2EDTA.

Mode o fAct ion of EDTA

Experiments were also carried out to assess the nature of inhibition by Na2EDTA. In these experiments, 30 ml nutrient broth containing either no Na2EDTA or 0.5%

Na2EDTA was inoculated with 1% inoculum of the active culture. One ml samples were withdrawn at 0, 8, and 24 h, diluted suitably and plated on TGY medium. The colonies were counted after 48 h incubation at 28~ It was observed that cfu numbers of nutrient broth without Na2EDTA increased from 1.5 x 106 to 1 x 108 m1-1 within 24 h at 28~ However, cfu numbers in Na2EDTA treated samples remained almost the same, the viable counts after 0 and 24 h being 8 x 105 and 9 x 105 ml d respective- ly. Thus, Na2EDTA did not kill the cells but only inhibited their multiplication.

The above studies are being extended to the preservation of whole shrimps.

References

Alur MD, Lewis NF, Kumta US (1971) Spoilage potential of predominant organisms and radiation survivors in fishery products. Indian J Exp Biol 9:48--52

Eisai Co. Ltd. (1966) Sterilization and preservation of meat products. Patent No. Fr, 1, 4 4 8 , 4 4 0 (C1. A 23b)

258 R. Chander and N.F. Lewis

Igarashi H, Zama K (1972) Preservation of edible sea-foods. Patent No. Japan. 71:19, 576 (c1. A 23b)

Levin RE (1967) The effectiveness of EDTA as a fish preservative. J Milk Food Technol 30:277-283

Love TD, Thompson MH (1965) Annual report Bureau of Commercial Fisheries Tech- nology Laboratory, Pascagoula, Miss. U.S. Dept. Interior, Circ. 251:15-23

Metcalf RH, Deibel RH (1972) Growth of Streptococcusfaecium in the presence of lysozyme. Infect Immun 6:178-183

Metcalf RH, Deibel RH (1973) Effect of lysozyme on enterococcal viability in low ionic environments. J Bacteriol 113:278-286

Repaske R (1958) Lysis of Gram negative organisms and the role of versene. Biochem Biophys Acta 30:225-232

Vanderzant C, Mrog E, Nickelson R (1970) Microbial flora of Gulf of Mexico and pond shrimp. J Milk Food Technol 33:346-350

Received October 3, 1979