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ISSN : 1225-4339(Print)
ISSN : 2287-4992(Online)
The Korean Journal of Food And Nutrition Vol.26 No.4 pp.678-684
DOI : https://doi.org/10.9799/ksfan.2013.26.4.678

저염 오징어 젓갈의 숙성 중 오징어 먹즙 첨가가 젖산균의 변화에 미치는 영향

오 성 천
대원대학교 제약식품계열

Influences of Squid Ink Added to Low Salt Fermented Squid on Its Changes in Lactic Acid Bacteria

Sung-Cheon Oh
Dept. of Food & Pharmacy, Daewon University College, Jecheon 390-702, Korea

Abstract

This study measured the change of lactic acid bacteria duringthe ripening fermentation process of low salt fermented squidwith no squid ink added. All study groups showed increase ofLeuconostoc and rapid growth of total plate count at thebeginning stage of ripening and the maximum microbial countshowed at the optimum stage of ripening which graduallyreduced after the optimum stage. It is believed that Lactobacillusoccupied the major part of the total plate count after theoptimum stage of the squid fermentation, and it was related tothe quality after the optimized ripening stage. Streptococcus andPediococcus were gradually increased until the optimum stageof the ripening, and then decreased rapidly. Yeasts were detectedin the middle stage of the fermentation and rapid increase wasshown after the last stage of the fermentation which suggeststhat yeasts participate in putrefaction of the low salt fermentedsquid. The change of lactic acid bacteria observed during theripening fermentation of low salt fermented squid with squidink added was that the total plate count increased until ripeningmiddle stage but showed a tendency to slightly reduce after themiddle stage. The length of time to reach the maximum valuewas longer than the no treatment groups. Among the lactic acidbacteria, Leuconostoc, Streptococcus and Pediococcus has increaseduntil the middle stage of the ripening while Lactobacillusconstantly increased to the end part of the ripening. Yeasts hadno increasing in the early ripening stage, but after middle ofthe ripening, it started to increase. That kind of tendency wassimilar to the case of no treatment groups. However, the amountof lactic acid bacteria tended to be less than no treatment groups.The tendency of decreasing number of all bacteria in low saltfermented squid with squid ink added shows squid ink restrictsthe growth of all bacteria.

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Introduction

 Fermented squid is a fermented seafood product which contains unique flavor caused by a reaction between protease produced by microorganisms and during the ripening stage. The various microorganisms in fermented seafood contribute to preservability as well as flavor and color of the product (Shinano et al. 1975).

 Research proved the proliferation distribution of Micrococcus and Staphylococcus using the microorganisms separated and identified from 10%, 15%, and 20% salt concentration fermented squid experimental groups and also determined Staphylococcus is a dominant strain in the ripening stage regardless of the salt concentration although higher salt concentration reduces the bacterial count in an extract (Mori et al. 1979; Mori et al. 1983).

 Study reported Bacillus, Corynebacterium, Lactobacillus, Moraxella, Micrococcus, Acinetobacter, Staphylococcus, Streptococcus, and extra have been homogenized from fermented squid and Staphylococcus aureus has been detected from fermented squid 4-7% of low salt concentration (Fujii et al. 1991; Fujii 1984; Matsubara et al. 1994).  

 Study reported that squid intestine contributes an important role in forming a special mycoflora in fermented squid (Yamazaki et al. 1992) and another study claims yeast or Pseudomonas are related with the ripening of fermented squid (Shinano et al. 1975; Hur et al. 1995). Study reports existence of lactic acid bacteria in fermented seafood including fermented squid. A fish sauce produced in Southeast Asia contains lactic acid, acetic acid, and pyroglutamic acid that result from lactic acid fermentation by acid forming microorganisms. This report also shows lactic acid bacteria are the main Bacillus during the ripening of fermented seafood. Shiokara which is Japanese fermented squid contains 106-107 amounts of Lactobacillus and fermented shrimp contains 102-104 of Lactobacillus in condition of 104-106 total plate counts (Itoh et al. 1985). Identification of lactic acid bacteria from low salted fermented squid shows Lactobacillus farciminis, Lactobacillus corniformis, Lactobacillus confusus and Lactobacillus plantarum and these 4 strains grow favorably at 25℃ (Morishita et al. 1995). During the ripening of 7% and 9% salt concentration fermented squid, Staphylococcus xylosus, Micrococcus varians, Pseudomonas diminula, Flavobacterium odoratum existing in fermented squid had been detected and homogenized and also counted their total plate count change (Kim et al. 1993; Kim et al. 1995). Studies are being conducted about the production of squid Sikhae which is another product (Kim et al. 1994; Lee et al. 1996). A trait of microorganisms related with fermented squid ripening is the existence of many micrococcus types but recent reports say that lactic acid bacteria have a big part in low slat fermented squid and reducing the amount of salt for health purposes is trending.  

 The purpose of this study is to contribute to an improvement in quality of fermented squid. The research analyzed the total bacterial count when 5%, 7% and 9% salt concentration fermented squid and 4% squid ink-added fermented squid are ripened at 20℃ and 10℃ and the change in lactic acid bacteria when 5% salt concentration squid as well as squid with 4% squid ink added are ripened at 10℃.  

Materials and Methods

1. Sample Preparation

 Squid (Todarodes pacificus) was used as a raw material for fermented squid. As Fig. 1, after defrosting the frozen squid at 4℃, meat excluding the intestines, head, legs and fins, which were removed, was used. Low salt fermented squid was made according to the Table 1 composition. Squid with no added ink and squid with 4% added ink were each ripened at 10℃ and 20℃. The ripened squid at 10℃ was used at an interval of one week and the squid at 20℃ was used at an interval of 4 days as analysis samples.

Fig. 1. Flow diagram of preparation of salt fermented squid.

Table 1. The compositions of salt squid samples with squid ink before fermented

2. Proximate Composition Analysis

 General ingredients were measured following the A.O.A.C. method. That is, high pressure drying method by heating for water, Micro-kjeldahl method for crude protein, Soxhlet extraction process for crude fat, and direct ashing method was used for ash.

3. Total Plate Count Analysis

 The sample fermented squid was moved into a blender cup and homogenized for 3 minutes at 15,000 rpm. Then it was diluted in 10 levels using 0.1% peptone water and after choosing 3 suitable levels the sample was incubated at 30℃ for 48 hours using the streak-plate method. Plate count agar (Difco Lab.) was used as a medium for counting total plate count number, as in Table 2.

Table 2. The contents of plate count agar medium for viable cell counts

4. Measurement of Number of Lactic Acid Bacteria

 10 g of sample from fermented product from the early stage to ripening was applied 90 ㎖ of sterilized distill water and ground aseptically. Then it was homogenized after being shaken for 10 minutes using a shaking incubator and diluted 10 times.

  This diluted solution was smeared in a medium depicted in Table 3 and cultured for 48 hours at 30℃. The total number of microorganisms was the number of colonies formed in plate count agar and the growing strains in the selected medium were counted (Lee et al. 1992; Cha et al. 1988; Shigeo & Toshio 1988).

Table 3. Compositions of media used in microbial studies (Unit: per liter)

 Lactobacillus was counted after acetic acid and sodium acetate were added to the LBS broth while PES broth was used for Leuconostoc. After adding 2,3,5-triphenyl tetrazolium chloride to m-enterococcus agar and reduction, if the colony appeared red, it was counted as Streptococcus and for a white colony, Pediococcus. Yeast was counted using potato dextrose agar.

5. Statistical Analysis

 For statistical processing, using Statistical Packages for Social Science (ver. 10.0, SPSS, Chicago, IL, USA), significant difference less than 5 percent (P<0.05) for the average value was researched. The study results indicated the average value and the standard deviation (Choi & Kim 2011).

Result and Discussion

1. Proximate Composition of Raw Squid

 General ingredients of the raw squid used in the study are according to Table 4. The raw squid’s water content, crude protein, crude fat, crude ash and carbohydrate content were each 78.0, 18.2, 1.2, 1.7, 0.8%. Because only salt was added to the intestine-removed squid during production of fermented squid, fat content is low and protein content is relatively high. This could be affected by the area and time when the squid was caught and this result is similar to that of Lee & Kim (2012).

Table 4. Proximate composition of raw squid (%)

2. Changes in Total Plate Count

 Fig. 2 shows the measurement of total plate count in low salt fermented squid of different salt concentrations ripened at 10℃. In the case of the 5% salt concentration sample, total plate count rapidly increased after he first week of ripening slowing down to a gradual increase until the fourth week when maximum total plate count was maintained at 108 (CFU/g). For the 9% salt concentration sample, total plat count showed a tendency to increase until the third week. After ward there was a small increase but mostly the total plate count maintained at 106-107 (CFU/g). Increase in salt concentration resulted in lower total plate count, meaning it inhibits growth of microorganisms.

Fig. 2. The viable cell counts of salt fermented squid during fermentation at 10℃.

 Fig. 3 shows total plate count in low salt fermented squid with 4% squid ink added ripened at 10℃. The squid ink added to low salt fermented squid resulted in lower total plate count, meaning squid ink inhibits growth of microorganisms.

Fig. 3. The viable cell counts of salt fermented squid with 4% squid ink during fermentation at 10℃.

 Fig. 4 shows total plate count in low salt fermented squid of different salt concentrations ripened at 20℃. In the 5% salt concentration sample total plate count rapidly increased on the fourth day, slowed down until the eighth day when it showed maximum total plate count of 108 (CFU/g) then decreased. In the 9% salt concentration sample the maximum total plate count showed at 107 (CFU/g) on the twelfth day of ripening.

Fig. 4. The viable cell counts of salt fermented squid during fermentation at 20℃.

 Fig. 5 shows the results of sensory evaluations of bacteria in low salt fermented squid with 4% squid ink added ripened at 20℃ carried out in 4 day intervals. In the 5% salt concentration sample the optimum stage of ripening was observed on the twelfth day. In the 9% salt concentration sample the maximum total plate count appeared on the eighth day but that number was low and it is assumed it cannot increase any larger.

Fig. 5. The viable cell counts of salt fermented squid with 4% squid ink during fermentation at 20℃.

 According to these results, in the without addition of the squid ink sample low salt concentration and high ripening temperature resulted in higher total plate count.

 However depending on the salt concentration the types of bacteria are largely different and therefore organically there will be differences.

3. Changes in Lactic Acid Bacteria

 Because fermented squid cannot be perfectly sterilized the quality of the squid depends on the microorganisms involved during the ripening process. The change in microflora when the 5% salt concentration sample was ripened at 10℃ is depicted in Fig. 6 and the change in the squid ink added sample is shown in Fig. 7.

Fig. 6. Changes in lactic acid bacteria of 5% salt fermented squid during fermentation at 10℃.

Fig. 7. Changes in lactic acid bacteria of 5% salt fermented squid with 4% squid ink during fermentation at 10℃.

 In all samples Leuconostoc rapidly increased in the beginning stage of ripening, forming an important part of the total plate count while maximum count was achieved at the optimum stage of ripening. In the case of the 5% salt concentration sample bacterial count increased quickly compared to the ink-added sample. Therefore squid ink is assumed to suppress the growth of microorganisms. Leuconostoc, which increased rapidly during the beginning stage, rapidly decreased during the optimum stage of ripening.

 Lactobacillus was detected in small amount in the beginning stage but increased rapidly after the optimum stage, forming most of the total plate count. This leads to the conclusion that it contributes to the quality of fermented squid after the optimum stage rather than the beginning stage.

 In the 5% salt concentration sample the total plate count reached 108 (CFU/g) in just three weeks and Streptococcus and Pediococcus increased to 107 (CFU/g) in five weeks. When decomposition occurs after the optimum stage yeast rapidly increases so it can be assumed that yeast is related to decomposition after optimum stage.

  In the case of the ink-added sample the change in microorganisms showed a similar course; however a delay in optimum stage could be observed. In all samples Leuconostoc rapidly increased during the beginning stage, forming an important part of the total bacterial count while in the middle stage Lactobacillus constituted as the prominent microflora.

  In a study for Japan’s representative fermented squid, Ikashiokora, Lactobacillus farciminis, which is a kind of lactic acid bacteria, was the most dominant bacteria during the 5℃ low temperature ripening process. In 25℃ high temperature ripening Lactobacillus confusus, Lactobacillus farciminis and Staphyllococcus epidermidis were the most dominant. The study claimed to have segregated Lactobacillus and that Lactobacillus is the most dominant bacteria (Morishita et al. 1995).

 Microorganisms such as Leuconostoc, Lactobacillus, Streptococcus and Pediococcus are lactic acid forming bacteria and therefore Gram positive bacteria. In this study the changes of these microorganisms were compared according to temperature and salt concentration. The reason these Gram positive bacteria increase in number during the beginning stage of ripening but decrease later is because the lactic acid formed by these bacteria causes the pH to decrease, restricting growth of bacteria.

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