Abstract

Shrimps are highly valued worldwide. However, they deteriorate rapidly after harvest except preserved or subjected to hazard analysis critical control point (HACCP). Therefore, this investigation was undertaken to determine the microbiological and physico-chemical [pH and trimethylamine (TMA)] characteristics of shrimps subjected to several critical control points (CCPs) including 100 ppm sodium metabisulphite (Na₂S₂O₅) alone or followed by 10 ppm calcium hypochlorite Ca[OCL]₂ before ambient (27-35°C) or refrigeration (4-6°C) storage. Also, shrimp types (whole, head or tail subjected to CCPs (iced or un-iced storage) wereanalysed for total viable counts (TVCs), coliforms, Staphylococcus spp.,Salmonella spp. and Vibrio spp. counts; pH and TMA contents. Significant (p<0.05) maximum TVCs of 1.8 x 108cfu/g occurred in tail samples subjected to Na₂S₂O₅ followed by Ca[OCL]₂ before 27-35°C storage. Significant different bacterial populations occurred with the un-iced whole shrimps showing maximum population (2.9 x 105cfu/g) of coliforms. Additionally, Staphylococcus spp. had the maximum count (8.2 x 103cfu/g) in un-iced whole samples while the lowest (7.8 x 101cfu/g) occurred in tail samples treated with Na₂S₂O₅ followed by Ca[OCL]₂ before 4-6°C storage. Variations in bacterial profiles were influenced by the CCPs resulting in diverse bacteria with iced head samples showing Bacillus spp.,E.coli, Pseudomonas spp., Salmonella spp. and Staphylococcus aureus while the others differed. Most bacterial pathogens occurred in tail samples subjected to CCPs before 27-35°C storage. Highest pH (7.85) and TMA (37.48mgN/100g) occurred in tail samples treated with Na2S2O5 followed by Ca[OCL]₂ before ambient storage. Significant positive correlation occurred between TVCs and coliforms (r = 0.9011) and others. However, pH and TMA showed negative or poor correlation against the different bacterial groups.Percentage frequency of bacterial occurrence differed. This study has demonstrated the importance of HACCP and the need to adopt its concept and application to enhance microbial safety of shrimps.

1. Introduction

Shrimps are important seafoods globally and have high commercial value in export/international trade ^{1, 2, 3}. However, the quality attributes of shrimps deteriorate soon after harvest leading to substantial economic losses ^{4, 5}.

Shrimps harvested from developing countries are usually by artisanal fishermen who are not equipped with refrigeration facilities. This often results in high level of post-harvest losses due to substantial perishability ^{6, 7}.

A number of factors including contamination of shrimp ecosystems, poor handling practices, lack of adequate storage facilities and inadequate preservative measures contribute considerably to shrimp losses ^{5, 8, 9}.

Application of sulphiting agents in seafoods is permitted in many countries at permissible levels ^{4, 10, 11} with the main purpose of minimizing or preventing melanosis (black spot formation). The most commonly used agent is sodium metabisulphite which has also shown inconsistent reduction in microbial populations ^{1, 11}.

Changes in the pH of shrimp under different storage conditions have been reported to be minimal ⁵ while increase in trimethylamine (TMA) content of shrimp occurred during storage under refrigeration temperature ^{1, 12}.

HACCP application is highly recommended for sustenance of seafood quality and safety enhancement. However, HACCP is rarely adopted by seafood handlers and industries as well as regulatory agencies especially in developing countries including Nigeria ^{13, 14}. More importantly, little or no work has focused attention on HACCP application for shrimp processing as a comprehensive approach to maintenance of shrimp quality after harvest ^{3, 7}.

The present work was therefore undertaken to determine the effects of HACCP concept on microbiological profiles and physico-chemical characteristics of shrimps based on the application of sodium metabisulphite (Na₂S₂O₅), calcium hypochlorite Ca[OCl]₂, headed versus tail portion and storage temperature as critical control points.

2. Materials and Methods

Freshly harvested shrimps (Penaeus notialis) (approximately 3kg) from Andoni River, Rivers State, Nigeria were purchased from seafood harvesters following prior arrangements. The samples ranged from 6-10g (with average weight of about 7g).

The shrimps were sorted into comparable sizes (approx. 7g each) and divided into two portions; one kept in ice-box (4-6°C) while the other in un-iced box. Figure 1 shows the critical control points as deemed appropriate in shrimp processing to improve microbial safety.

On arrival at the laboratory, the samples (iced and un-iced) were aseptically de-headed respectively into head and abdominal (tail) portions and analyzed for microbiological and physico-chemical characteristics.

The fresh abdominal (tail) portions (being more commercially valued and acceptable) were subjected to various critical control points (CCPs) or treatments as shown in Figure 1.

Following these CCPs/treatments, the samples were analyzed for microbiological and physico-chemical attributes.

Serial dilutions were prepared by blending 25g of pooled samples in 225ml 0.1% peptone water using a stomacher (model BA 6021, Seward Medical, London, UK).Further 10-fold dilutions were prepared and spread-plated (0.1ml aliquot) in triplicate on surface-dried plate count agar, MacConkey agar, Mannitol salt agar, Thiosulphite-citrate-bile-sucrose agar and Salmonella-Shigella agar and incubated at 37°C for18-24hr. The plates were then examined for colonial growth and enumeration astotal viable counts, coliforms, Staphylococcus spp., Vibrio spp and Salmonella- spp counts. All the culture media used were products of Titan Biotech. Ltd., India.

Typical colonies were randomly picked from plates showing 30-300 colonies, purified and identified based on cultural, morphological and biochemical characteristics. The identification procedure included colonial and cellular morphological features coupled with biochemical characteristics (IMViC, triple sugar iron agar, catalase, oxidase, sugar fermentation:glucose, lactose, sucrose, mannitol ^{15, 16, 17}.

pH and Trimethylamine (TMA)

2.5.1. The pH of the pooled 10g samples of the respectively treated samples were determined after blending in 20ml distilled water (1:2 ratio) ¹⁸ using a calibrated pH meter (model Jenco 6177, USA).

2.5.2. The TMA contents of the respective triplicate samples were determined as described by Malle and Poumeyrol ¹⁹. The determination involved use of Kjedahl distillation unit 2100 (Foss, Sweden).

The obtained data were analyzed using Analysis of variance (ANOVA) software of SPSS version 15 and the significance of the mean differences determined at p<0.05.

3. Results

The microbiological qualities of the shrimps varied due to the critical control points/treatments (Table 1). The maximum total viable count (1.8 x 10⁸cfu/g) occurred in tail samples stored at ambient temperature following 100 ppm Na₂S₂O₅ and 10ppm Ca[OCl]₂ treatment which was significantly different from the other samples (Table 1). Additionally, the other samples differed significantly from each other but those treated with Na₂S₂O₅ and Ca[OCl]₂ before refrigeration temperature showed the lowest total viable counts (Table 1). Coliform counts exhibited similar trends but un-iced samples on arrival in the laboratory had the highest significant population of 2.9 x 10⁵ cfu/g (Table 1). Similarly, Staphylococcus spp counts showed significant maximum population of 8.2 x 10³cfu/g in un-iced whole samples while the lowest count (7.8 x 10¹ cfu/g) was observed in tail samples treated with Na₂S₂O₅and Ca[OCL]₂prior to refrigeration storage (Table 1). Less significant variations in Vibriospp counts occurred as affected by the critical control points but samples stored at ambient temperature had the highest population of 2.2x10⁵cfu/g (Table 1). Similarly, maximum Salmonella spp. population (3.3 x 10⁵cfu/g) was observed in tail sample subjected to Na₂S₂O₅ treatment followed by Ca[OCl]₂ treatment before storage at ambient temperature. However, un-iced whole shrimp sample, iced whole shrimp sample and tail sample dipped in tap water (control) and drip-dried showed no significant (p<0.05) difference among these three treatments (Table 1).

Table 2 shows the variety of microorganisms isolated from the shrimp samples as influenced by the critical control points/treatments. Maximum of five different bacterial genera were isolated from iced head sample, tail sample treated with 100ppm sodium metabisulphite alone, tail sample treated with 100ppm Na₂S₂O₅ and 10ppm Ca[OCl]₂ and tail sample subjected to 100ppm sodium metabisulphite and 10ppm calcium hypochlorite prior to refrigeration storage (Table 2). However, control sample dipped in tap water showed six bacterial genera (Table 2). But four different bacterial genera were isolated from the other samples.Of the bacterial isolates, most pathogens (Bacillusspp., E. coli, Salmonella spp., Staphylococcus spp.and Vibrio spp.) occurred in control samples, un-iced whole samples, iced head sample, tail samples treated with 100pm sodium metabisulphitealone, tail samples treated with 100ppm Na₂S₂O₅and 10ppm Ca[OCl]₂ and tail samples treated with 100 ppm sodium metabisulphite followed by calcium hypochlorite prior to ambient temperature storage (Table 2). In addition, Pseudomonas spp.were mostly isolated from iced or refrigerated samples irrespective of sample type (whole, head and tail) (Table 2).

The pH values and TMA contents of the shrimps as influenced by the critical control points/treatments are shown in Table 3. The tail portion subjected to 100ppm sodium metabisulphite followed by 10ppm calcium hypochlorite prior to ambient temperature storage and that dipped in tap water (control) showed the highest significant pH values (Table 3). Significantly (p<0.05) higher pH value occurred in head portion compared with tail portions but the lowest pH value was observed in tail portions treated with 100 ppm sodium metabisulphite and 10 ppm calcium hypochlorite (Table 3).

The tail portion treated with 100ppm sodium metabisulphite and 10ppm calcium hypochlorite prior to ambient temperature storage showed the highest TMA content (37.48 mgN/100g) which was followed by the control sample (Table 3). But un-iced whole sample and iced head sample had comparable TMA contents while the lowest TMA content (5.49 mgN/100g) occurred in shrimps treated with 100ppm sodium metabisulphite alone (Table 3).

Each value represents the mean of triplicate determinations. Mean values in columns under pH and TMA having different letters are significantly (p<0.05) different.

The correlation values of the different variables and the levels of significance are presented in Table 4. Highly significant positive correlation occurred between total viable counts and coliform counts (r=0.9011), between coliform counts and Salmonella spp. population (r = 0.9692), between Vibrio counts and Salmonella spp. population (r=0.9732) and total viable counts versus Salmonella spp. population (r = 0.7803) (Table 4). However, total viable counts versus Vibrio spp. counts showed weak positive correlation (r = 0.6516) while non-significant negative correlation occurred between pH and total viable counts (r=-0.3406), between pH and coliforms (r = -0.2867) and between TMA and total viable counts (r = - 0.3406). Similarly, a weak non-significant positive correlation (r = 0.3361) was observed between TMA and coliform counts (Table 4).

The percentage frequency of occurrence of bacteria isolated from un-iced whole fresh shrimps and iced whole fresh samples is shown in Figure 2a and Figure 2b respectively.Whereas five bacterial genera were isolated from un-iced samples, four bacterial genera occurred in iced samples with E. colibeing the most predominant (26%) in un-iced samples followed by Salmonella spp. (22%); Staphylococcus spp. was least prevalent (Figure 2a).

In contrast, Pseudomonas spp. was the most prevalent (30%) and Proteus spp.was least dominant (18%) in iced whole fresh shrimps (Figure 2b).

The iced head portion of the shrimps showed Pseudomonas spp. having the highest frequency of occurrence (28%) followed by Bacillusspp. (24%) while the least was Salmonella spp. (15%) (Figure 3a).

Conversely, iced tail portion had Bacillus spp. as the most dominant (30%) and E.coli as the least prevalent (11%). Tail portion of shrimps subjected to critical control point using 100ppm sodium metabisulphitealone showed Bacillus spp. havingmaximum frequency of occurrence (33%) while the lowest frequency of occurrence was 12% obtained forVibrio spp. (12%) (Figure 3b).

On the other hand, tail portion treated with 100ppm sodium metabisulphite and followed by 10ppm calcium hypochlorite had much higher Staphylococcus spp. (28%) compared to that treated only with 100ppm sodium metabisulphite (Figure 4 and Figure 5).

In addition, no Vibrio spp. was observed in Figure 5. The tail portion subjected to critical control point involving tap water (control) had six bacterial isolates with E.coli showing the maximum frequency of occurence (24%) followed by Bacillus spp. (20%) and Staphylococcus spp. (10%) (Figure 6).

Figure 7a shows percentage frequency of occurrence of bacteria isolated from tail portion treated with 100ppm sodium metabisulphite and 10ppm calcium hypochlorite before refrigeration storage for 48hr. Much higher percentage of Pseudomonas spp. occurred (34%) followed by E. coli (22%) and the lowest,Staphylococcus spp. (11%) (Figure 7a).

But fewer bacterial genera were observed in tail sample treated with 100ppm sodium metabisulphitefollowed by 10ppm calcium hypochlorite before ambient temperature storage (27-35°C) for 48hr (Figure 7b) which was dominated by Bacillus spp. (38%) and Staphylococcus spp. (25%)(Figure 7b).

4. Discussion

Critical control points (CCPs) or control measures applied in food production systems are often aimed at minimizing or preventing hazards in foods to enhance food safety ^{4, 13, 20}. Storage temperature of food and use of preservatives are among the critical variables associated with microbial growth, proliferation and safety in foods ^{8, 18, 21}.

The present results (Table 1) have clearly shown the importance of storage temperature as a critical control point in shrimp production process where samples stored at ambient temperature exhibited maximum total viable counts (TVCs) in spite of treatment with Na₂S₂O₅ followed by Ca[OC]₂ (Table 1). Similarly, un-iced whole shrimps compared with iced whole shrimp had significantly much higher microbial population (1.8x10⁶ cfu/g)than the latter (Table 1). The variations in the TVCs as influenced by the respective critical control points (Table 1) underscore the need for application of CCPs in shrimp processing. For example, the application of sodiummetabisulphite alone was beneficial but sequential application of calcium hypochlorite was significantly more effective in reduction of TVCs count of and other microbial groups (Table 1). This gives credence to the report by Pyle and Koburger ¹¹ that the antimicrobial effects of sodium metabisulphite treatment were dependent on order of application such as the sequential treatment of shrimp with bisulphite followed by hypochlorite which led to greater microbial reduction over either treatment alone (Table 1). The occurrence of similar trends among the different bacterial groups (Coliforms, Staphylococcus spp.,Vibrio spp. and Salmonella spp.) suggest the effects of CCPs where gram negative bacteria predominated in samples stored at low temperature or subjected to iced-storage as earlier reported ^{1, 5, 8}.

The high microbial populations observed in the un-iced and ambient temperature stored samples (Table 1) corroborate the findings by other investigators who showed highest pathogenic microbial loads during the summer ²². Apparently, based on the TVCs safety limit of 10⁵cfu/g of shellfish ^{6, 23, 24}. un-iced whole shrimp, tail sample subjected to tap water (control) and sample treated with 100pm Na₂S₂O₅ followed by 10ppm Ca[OCL]₂ prior to ambient temperature storage (Table 1) are therefore microbiologically unsafe having exceeded the limit. Thus, the application of CCPs involving low temperature and/or preservatives enhanced the microbial safety of the samples.

The isolation of fewer bacterial genera (five) from sodium metabisulphite and calcium hypochlorite treated samples is indicative of their inhibitory effects but such impacts were negligible in ambient temperature stored samples (Table 2). Thus, application of adequate CCPs should always be recommended as evidenced by the diversity of bacterial genera (six) in control sample (Table 2). In addition, whereas fewer bacterial isolates were observed in the ambient temperature stored samples (Table 2), virtually all of these microorganisms isolated from such samples were pathogens commonly associated with mesophilic growth temperature ⁴ (Table 2).

The pH of seafoods serves as an indicator of quality ^{1, 5}. In the present work, the occurrence of highest pH values in control and 100ppm sodiummetabisulphiteand 10ppm calcium hypochlorite treated samples prior to ambient temperature storage (Table 3) clearly reflect the impacts of CCPs in which the maximum pH of such samples is indicative of spoilage (Table 3). These results corroborate some earlier findings which showed that pH of shrimps is a useful indicator of freshness or spoilage ^{1, 12}. It is likely that the autocatalytic phenomenon involving bisulphite reaction with hypochlorite must have led to formation of hydrogen ions which probably reduced the pH and enhanced the antimicrobial effects ¹¹.

TMA is a known product of bacterial activity which is used as an index of seafood quality ^{25, 26}. The significantly higher TMA contents observed in the samples subjected to 100ppm sodium metabisulphite followed by 10ppm calcium hypochlorite before ambient temperature storage or control sample (Table 3) clearly indicate the impacts of microbial activity as influenced by storage temperature or lack of preservative (ie control) (Table 3).It is therefore evident that the beneficial effects of sodium metabisulphite and calcium hypochlorite coupled with low temperature are reflected by the low TMA contents (Table 3). These results are in agreement with some other findings involving use of ice-storage of shrimps ¹. However, the significantly high TMA contents (Table 3) that occurred in control and un-iced whole and iced head samples are indicative of the importance of sample portion/type ⁶ and CCPs in relation to shrimp deterioration or shelf life extension ^{1, 9, 12}.

Thus, based on the range of 10-15mgN TMA/100g for seafood spoilage-detection and unacceptability ^{4, 12, 18}, it is therefore evident that virtually all the samples except three, namely; (i) those subjected to 100ppm sodium metabisulphitealone (ii) those subjected to 100ppm sodium metabisulphite followed by 10ppm calcium hypochlorite prior to refrigeration storage and (iii) whole shrimp subjected to iced storage were considered unacceptable having exceeded the limit of 15mg TMA/100g (Table 3).

Correlation analysis showed that total viable counts were significantly positively related to the coliforms and Salmonella spp.counts respectively. Such a relationship was also established between coliforms and Salmonella spp. counts as well as between Vibrio spp. and Salmonella spp. populations (Table 4). Thus, each of these microbial groups may be used as predictor of the other. In contrast, the physico-chemical parameters (pH and TMA) either correlated poorly or negatively against the microbial groups (Table 4) which suggest that they are poor indices of microbial predictability in these samples probably due to the influence of the various treatments.

The differential percentage frequency of bacterial occurrence between un-iced and iced whole shrimp samples clearly suggests the effects of storage temperature since more bacterial diversity was observed in the samples exposed to high ambient temperature [27-35°C] with particularly Bacillus spp. and Staphylococcus spp. being most dominant which probably may be attributed to their mesophilic characteristics ^{4, 15}. On the other hand, the dominance of Pseudomonas spp. in iced whole samples (Figure 2b) confirms their proliferation under low temperature storage conditions as previously reported ^{4, 18}. Whereas the bacterial distribution (five) is comparable between the two sample portions, the head portion was dominated by Bacillus spp. while Staphylococcus spp.were most prevalent in the tail portion (Figure 3a and b). This differential bacterial profile could be attributed to tissue anatomical structure and exposure as well as bacterial ability to survive under different conditions since Bacillus spp. are spore-formers and Staphylococcus spp. are more associated with aerial and food processing contamination ^{4, 6}. Similarly, the predominance of Bacillus spp. and Staphylococcus spp. in samples treated with the preservatives suggeststheir tolerance as spore-formers and the sensitivity of gram-negativeasporogenous bacterial genera (Figure 4 and 5). Evidently, the occurrence of maximum bacterial diversity of six genera (mostly pathogens)in control samples indicates the potential hazards posed by such samples to consumers (Figure 6) hence, CCPs are highly recommended. Application of low storage temperature is highly advisable but often, not available in developing countries including Nigeria due to inadequate (erratic) power supply ^{18, 27}. Nevertheless, samples stored under refrigeration exhibited substantial prevalence of Pseudomonas spp. which are known to play a major role in spoilage of such seafoods ^{3, 4}.

On the contrary, the occurrence of fewer bacterial genera in samples subjected tosodium metabisulphite and calcium hypochlorite before ambient temperature storage (Figure 7b) but dominated by Bacillus spp. (38%) and followed by Staphylococcus spp. clearly underscores the critical importance of storage temperature since the effects of high ambient temperature (27-35°C) outweighed the benefits of the preservatives (Figure 7b). These results clearly indicate the essence of hurdle technology and application of HACCPconcept in the seafood industry.

5. Conclusions

Application of critical control points (CCPs) in shrimp processing has shown that use of sodium metabisulphite followed by calcium hypochlorite significantly improved microbial safety of shrimps. Further enhancement of shrimp safety was achieved when such preserved samples were subjected to iced or refrigeration storage. However, ambient temperature storage of shrimps impacted negatively with respect to microbial and physico-chemical parameters in spite of use of sodium metabisulphite and calcium hypochlorite as CCPs. Additionally, the prevalence of pathogens in samples subjected to ambient temperature storage is a major concern. Whereas total viable counts correlated positively with other bacterial groups, poor or negative correlation was observed between the physico-chemical parameters (pH and TMA) and the microbial groups. Therefore, they are poor predictors of microbial quality/safety in these samples. This study has clearly demonstrated the benefits of application of sodium metabisulphite followed by calcium hypochlorite as critical control points in shrimp processing. But these effects waned when such samples were subjected to ambient temperature storage. It is therefore evident that application of HACCP concept with focus on various relevant control measures is necessary to achieve microbiologically safe food products. Thus, this study has provided information (not currently available) on the concept and application of HACCP to seafood harvesters, processors and regulatory agencies such that its adoption would enhance microbial safety of shrimps and other food products.

Acknowledgements

Tertiary Education Trust Fund (TetFund) is gratefully acknowledged for the sponsorship of this research project (TETFUND/DESS/UNI/P-HARCOURT/IBR/2016/VOL.1) to Prof. B.J.O Efiuvwevwere, the Lead Researcher of the team.

Competing Interests

Authors have declared that no competing interests exist.

References