Assessment of the inhibitory effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate on Staphylococcus aureus growth and staphylococcal enterotoxin A production in cooked pork sausage using a predictive growth model

Lu LinJie Yun HuYi WuMin ChenJie OuWei Ling Yn

aShanghai Food Research Institute,Shanghai 200235,China

bShanghai Municipal Center for Disease Control and Prevention,Shanghai 200336,China

cShanghai Ocean University,Shanghai 201306,China

Abstract

Keywords:Staphylococcus aureus;Staphylococcal enterotoxin A;Cooked pork sausage;Preservative;Sodium lactate

1.Introduction

Staphylococcal food poisoning(SFP)is caused by staphylococcal enterotoxins(SEs)produced during massive growth ofStaphylococcus aureusin food[1–3].SFP is one of the most common foodborne illnesses in the United States[4],and it is also very common in China[5–7].S.aureushas the ability to grow and produce SEs over an extensive range of pH,water activity(Aw),sodium chloride concentration,and temperature,leading to intoxication from an equally extensive range of foods[1].SFP is often associated with protein-rich food,such as meat and dairy products,which may be subject to extensive manual handling,inadequate heating,or inappropriate storage[8].

Being a type of popular meat product,cooked pork sausage has shown a fast growing market in China[9].It is produced by blending minced raw pork meat with other ingredients,mix-ing and cutting,filling into a casing,and then cooking using water tank.Like most of the cooked meat products sold at delis in China,cooked pork sausage was found to be stored at delis without packaging,thereby making it susceptible to contamination by microorganisms,including pathogens such asS.aureus,from the environment during storage and distribution;moreover,its high water activity and rich nutrition encourageS.aureusto grow and produce SEs,and even to cause SFP[10,11].Several studies on food safety monitoring conducted in China[12–14]have indicated thatS.aureuswas one of the most common foodborne pathogens associated with cooked meat products.Therefore,controlling the growth ofS.aureusis an important issue for ensuring the safety of cooked meat products.Indeed,inhibitingS.aureusgrowth and SEs production in cooked meat sausage will be beneficial,and one possible solution is to identify effective preservatives that can be added into the product,so that whenever the product is contaminated withS.aureus,the growth could be effectively inhibited or at least the production of SEs could be delayed.Thus,the safety of the cooked pork sausage could be significantly improved.

According to the Chinese National Food Safety Standard for Uses of Food Additives(GB2760-2014),sodium nitrite,nisin, potassium sorbate, and sodium lactate are legally approved to be added into cooked meat products as preservatives.The maximum levels of sodium nitrite,nisin,and potassium sorbate allowed to be added into meat products are 0.15,0.5,and 0.15g/kg,respectively,while there is no use limitation on the level of sodium lactate.The United States Department of Agriculture and Food Safety and Inspection Service allows the use of sodium lactate as a preservative in processed meat products,and the maximum level is 48g/kg[15].Several studies have reported about the effects of preservatives on the growth ofS.aureus[16–20].However,the results of these studies were sometimes inconsistent,as the antimicrobial activity is affected by a number of environmental factors,including pH,inoculum size,and interaction with food components.Moreover,as SEs are considered as the causative agents of SFP,the detection of SEs is essential for assessing the risk of SFP;however,till date,only a few studies have reported about the applicability of SEs detection for assessing the effect of preservatives againstS.aureusgrowth.

The growth of microorganisms is known to be greatly influenced by environmental factors.Food preservatives could be considered as a type of environmental factor that affects the growth of microorganisms[21].Since the microbial predictive model can be used to assess the impact of food handling and storage conditions on the risk of foodborne pathogens in food and public health[22],we assumed that it could also be used to quantify the effects of preservative factors on the growth ofS.aureus.The objective of this study was to assess the inhibitory effects of four preservatives on the growth ofS.aureusand SEs production in cooked pork sausage;anS.aureusstrain producing staphylococcal enterotoxin A(SEA),the most common type of SEs found in food products[3,23],was chosen to be inoculated into the prepared cooked pork sausage containing one of the preservatives.The growth predictive model ofS.aureusin cooked pork sausage coupled with the detection of SEA was used to estimate the inhibitory effects of the four preservatives on the growth ofS.aureusand SEA production.The results from this study may be beneficial for manufacturers to formulate cooked pork sausages that limit the ability ofS.aureusto produce SEs,thereby reducing the risk of SFP.

2.Materials and methods

2.1.S.aureus strain and preparation of inoculum

S.aureusstrain(SA14966)was preserved in Shanghai Food Research Institute;this strain is known to produce SEA.To prepare the inoculum,0.1mL of thawed bacteria was transferred into 50mL of Luria–Bertani(LB)medium and were grown to the desired cell density of 8.0–9.0 log CFU/mL in a thermostatic oscillation incubator at 100rpm at 37°C for 18–20h.Single colonies ofS.aureuswere obtained from plate streaking,which were incubated at 37°C for 24h.Following the method of Peter and Robert[18],a loopful of bacteria was picked from a single colony,transferred into 50mL of LB medium,and incubated in a thermostatic oscillation incubator at 100rpm at 37°C for 18–20h.Then,the bacteria were centrifuged at 10,000rpm at 4°C for 20min and washed with sterile 0.1mol/L potassium phosphate buffer(pH 7)solution two times and diluted to the desired density of 2.0–3.0 log CFU/mL.Two times’washing guarantees that the resulting solution contains onlyS.aureuscells,without carrying SEA.

2.2.Preparation of samples with different preservatives and the experimental design

The cooked pork sausage samples were prepared based on regular process and formulation;the ingredients of the samples consist of lean and fat pork meat,soy protein,starch,and sodium tripolyphosphate.

In this study,two steps were designed to estimate the effects of the four preservatives againstS.aureusgrowth and SEA production.First,five cooked pork sausage samples were prepared according to the mentioned process and formulation,of which four samples contained maximum levels of 0.15g/kg sodium nitrite,0.5g/kg nisin,0.15g/kg potassium sorbate,and 48g/kg sodium lactate,respectively,and one sample contained no preservative(blank).Second,to better understand the inhibitory effects of the preservatives under various concentrations,additional cooked pork sausage samples containing the preservative that showed a significant inhibitory effect in the first step were prepared,some of them containing full,half,quarter,and eighth concentration of the maximum level of the selected preservative,and one sample containing no preservative(blank).

After cooking and cooling,the pH and Awvalues of the samples were tested using a pH meter(Delta 320,Mettler Toleddo,China)and an Awmeter(HygroLab2,Rotronic,Switzerland),respectively.

2.3.Inoculation,plate counting of S.aureus,and detection of SEA

The prepared sausage samples were divided into small blocks of about 50g and then immersed intoS.aureussolutions with bacterial cell concentrations of 2.0–3.0 log CFU/mL for about 5min.The inoculated samples were then packaged aseptically and stored at 37°C.The samples were analyzed in triplicate every 3h to count the number of colonies by 3M PetrifilmTMStaph Express Count Plate and expressed as log CFU/g.The temperature 37°C was chosen in this study because this is the optimum temperature forS.aureusgrowth by which the optimal preservative could be identified effectively.

For the detection of SEA in the cooked pork sausage samples,a Mini VIDAS automatic fluorescent ELISA test system(BioMérieux,France)and a VIDAS?staphylococcal enterotoxin II(SET2)kit(BioMérieux,France)as a detection kit were used.This measurement system uses the principle of enzyme-linked fluorescent assay(ELFA)using polyclonal anti-enterotoxin antibody.Results are expressed as relative fluorescence value(RFV),and TV indicates the RFV of the tes?t solution divided by the RFV of the standard in the VIDAS Staph enterotoxin SET kit.A test solution with a TV value of≥0.13 is considered positive.The detection limit is at 0.5ng/g of SEA.

Three replicates of both plate counting and SEA detection were performed for each condition.The average values and the standard deviations of the transformed values were then calculated.

2.4.Modeling the growth of S.aureus

The primary model is used to primarily describe the relationship between the microbial biomass changes with time under the condition of a specific environment or a culture medium[24,25].The modified Gompertz model was used in this study,and the results were used to estimate the inhibitory effects.The function-modified Gompertz model[26]is as follows:

N(t)represents the population of the growth of microorganisms at a particular time(log CFU/g),N0represents the model fitting for the initial microbial population(log CFU/g),A represents the difference between the maximal population and the initial population(log CFU/g),μ represents the maximal rate[(log CFU/g)/h],and λ represents the lag phase(h).

2.5.Statistical analysis

Microbial counts were expressed as log values for statistical analysis.The influence of the preservative conditions on the growth parameters(μ,λ)was evaluated through an analysis of variance(ANOVA),and post hoc tests(i.e.,Duncan’s test)were used to determine the homogeneous groups(p＜0.05)using IBM?SPSS?Statistics version 19.0.0.

3.Results

3.1.Technological profile of the prepared samples

Results of the testing showed that all the samples had Awvalues ranging from 0.935 to 0.950 and pH values ranging from 6.04 to 6.25(Table 1).All these ranges were optimal forS.aureusgrowth and SE production[27].

Table 1Awand pH values of the prepared samples.

3.2.Effects of the four preservatives against S.aureus growth and SE production

The initial concentration ofS.aureusin all the samples was about 2–3 log CFU/g.After 3.9h of the lag phase(λ),the blank sample reached the logarithmic phase;the concentration ofS.aureusat the stationary phase was 9.2 log CFU/g.Compared with the blank sample,the lag phase(λ)of the samples containing sodium nitrite,nisin,and potassium sorbate extended for 2–3h,and the concentration ofS.aureusat the stationary phase of the samples containing sodium nitrite, nisin, or potassium sorbate was about 8.7–9.2 log CFU/g,with no significant difference between that of the blank sample.However,the lag phase(λ)of the sample containing sodium lactate extended for 14h,and the concentration ofS.aureusat the stationary phase of the sample containing sodium lactate was only 4.7 log CFU/g,which was significantly lower than that of the blank sample(Fig.1).

Fig.1.(a–e)The growth of S.aureus and SEA production in cooked pork sausage under various preservative conditions.

The lag phase(λ)is the time required for the cell to adapt to a new environment[28].The results showed that the lag phase(λ)of all the samples containing the preservatives was longer than that of the blank,while the lag phase(λ)of the samples containing sodium lactate was much longer than that of the samples containing other preservatives, indicating that the inhibitory effect of sodium lactate was more significant than that of other preservatives.This result was also confirmed by the difference in the concentration ofS.aureusat the stationary phase.

According to the ELFA results,S.aureusproduced detectable SEA in the blank sample after 18h of storage at 37°C,and SEA was also detected in the samples containing sodium nitrite,nisin,and potassium sorbate after 15–18h,with no difference between that of the blank sample.However,no SEA was detected in the samples containing sodium lactate during 36h(Fig.1).

Based on the results of this experiment, it could be concluded that sodium lactate could significantly inhibitS.aureusgrowth and SEA production in the cooked pork sausage samples.Meanwhile,in contrast,sodium nitrite,nisin,and potassium sorbate had limited effects against the growth ofS.aureusand had no effect against SEA production(Fig.1,Table 2).

Table 2The results of the modeling by modified Gompertz model and the time when SEA could be detected under the maximum level of each preservative.

3.3.The inhibitory effect of sodium lactate at various concentrations

The results described in Section 3.2 show that sodium lactate had a significant inhibitory effect onS.aureusgrowth and SEA production at the maximum concentration.To understand its inhibitory effect at various concentrations,five cooked pork sausage samples were prepared with the same process and for-mulation,of which four samples contained sodium lactate at concentrations of 48,24,12,and 6g/kg,respectively,and one sample contained no preservative(blank).The prepared samples were then inoculated withS.aureusand stored at 37°C.Counting of theS.aureuscolonies and detection of SEA were carried out as mentioned in Section 2.3.

After 3.8h of the lag phase(λ),the blank sample reached the logarithmic phase;the concentration ofS.aureusin the stationary phase was 9.2 log CFU/g.Compared with the blank sample,the lag phase(λ)of the samples containing 6,12,and 24g/kg of sodium lactate extended for 1–3h.The concentrations ofS.aureusin the samples containing 6,12,and 24g/kg of sodium lactate at the stationary phase were 9.0,8.4,and 7.8 log CFU/g,respectively,which showed a decreasing trend.Meanwhile,the lag phase(λ)of the sample containing 48g/kg sodium lactate extended for 14h,and its concentration ofS.aureusin the stationary phase was only 5.0 log CFU/g,which was significantly lower than that of samples with other concentrations of sodium lactate.

According to the ELFA results,S.aureusproduced detectable SEA in the blank sample after 18h of storage at 37°C,and SEA was also detected in the samples containing 6 and 12g/kg of sodium lactate after 18h,which were similar to that of the blank sample.However,SEA could be detected in the sample containing 24g/kg sodium lactate after 24h,and no SEA was detected in the sample containing 48g/kg sodium lactate during 36h(Fig.2,Table 3).

Fig.2.(a–e)The growth of S.aureus and SEA production in cooked pork sausage under various concentrations of sodium lactate.

Table 3The results of the modeling by modified Gompertz model and the time when SEA could be detected under various levels of sodium lactate.

The results of this experiment indicate that the inhibitory effect on the growth ofS.aureusand SEA production appeared to be invalid when the sausage samples contained 6 and 12g/kg of sodium lactate.The inhibitory effect became valid only when the sodium lactate concentration in the sausage sample was increased to 24g/kg.

4.Discussion

In the present study,the primary predictive model was used to describe the growth ofS.aureusunder the conditions of different types of preservatives.Compared with the method of minimum inhibitory concentration(MIC)using culture media,the method of primary predictive model could be used to describe the growth ofS.aureusin food matrices more intuitively and actually.Furthermore,the detection of SEA in this study effectively confirmed the inhibitory effect of the preservatives onS.aureusgrowth.

Several studies analyzing the effects of preservatives on the growth ofS.aureushave indicated that the antimicrobial activity is affected by numerous environmental factors,including pH,inoculum size,and interaction with food components.In the present study,real cooked sausage samples were used as media instead of laboratory culture media,due to which the obtained results could be considered more reliable for practical use.It was observed thatS.aureusin the cooked pork sausage was well tolerant to sodium nitrite,nisin,and potassium sorbate,even under their maximum concentrations;however,the growth ofS.aureusand SEA production could be significantly inhibited under the presence of sodium lactate.

Sodium nitrite has been used in meat curing for several centuries.The primary effect of sodium nitrite as an antimicrobial agent is inhibition of the growth and toxin production ofClostridium botulinumin cured meats.In association with other components in the curing mix,such as salt,ascorbate,erythorbate,and pH,nitrite exerts a concentration-dependent antimicrobial effect on the outgrowth of spores fromC.botulinumand other clostridia.Nitrite has been shown to have variable effects on microorganisms other thanC.botulinum;the growth ofC.perfringens,Listeria monocytogenes,andSalmonellasp could also be inhibited by sodium nitrite at concentrations ranging from 0.2 to 0.4g/kg[29–31].However,till date,no research has reported about the significant effect of sodium nitrite against the growth ofS.aureus,which has also been confirmed in this study.

Organic acids are commonly used by food manufacturers as antimicrobial preservatives in a variety of food products.Compared with other organic acids,the antimicrobial activity of potassium sorbate is the highest in the undissociated state,so that the pH of the food approaching neutrality will result in the reduction of antimicrobial activity.Larocco et al.[32]demonstrated no inhibitory or harmful effects onS.aureusMF-31with potassium sorbate(0.3%)or sorbate in combination with salt at pH 6.3.In addition,Teng et al.[33]reported that Gram-negative bacteria could be more effectively inhibited by potassium sorbate than Gram-positive bacteria; the structural differences in the cell wall between the Gram-positive and Gram-negative bacteria may be the primary reason for the difference in tolerance to potassium sorbate.Hence,the weak inhibitory effect of potassium sorbate on the growth ofS.aureuscould be easily explained as it is a Gram-positive bacterium.

Nisin is an antibacterial multipeptide produced by a certain strain ofLactococcus lactis.It is a bacteriocin generally recognized as safe(GRAS)by the U.S.Food&Drug Administration and the World Health Organization since 1969[34].Nisin by itself has a narrow spectrum affecting only Gram-positive bacteria,includingAlicyclobacillus,Bacillus,Clostridium,Desulfotomaculum,Enterococcus,Lactobacillus,Leuconostoc,Listeria,Pediococcus,Staphylococcus,andSporolactobacillus.Laukova[35]found that several strains ofStaphylococcus(includingS.aureus)were sensitive to nisin.However,nisin activity is affected by a number of environmental factors,including the presence of food components such as lipids and proteins[36].Relevant studies[37–39]have shown that nisin exhibits strong inhibitory effect againstS.aureusin skimmed milk but has a weak effect in whole milk.This was probably due to the binding of nisin to fat globules[40];this binding could be overcome by surface application,including immersion[41],spray[42],and coating[43]with nisin.However,these methods appear to be inapplicable for industrial use.In this study,the combined presence of lipids and proteins in the meat and the subsequent heating reduced the antimicrobial activity of nisin,indicating that nisin mixed into the cooked pork sausage could not effectively inhibitS.aureusgrowth and SEA production.

Sodium lactate that has been used as a preservative in several fermented dairy,vegetable,and meat products can inhibit the growth of several types of spoilage bacteria and pathogenic bacteria[44]. Like other organic acids, the undissociated form plays an important role in its effectiveness.However,dissociated lactic acid could also be considered as an antibacterial functional group[45].It influences the formation of the electrochemical proton gradient,which plays an important role in the metabolism of microorganisms.Due to the formation of the electrochemical proton gradient,the microbial needs to consume more energy,and its energy deficiency affects its growth and reproduction[44].Wit et al.[46]found that 5% sodium lactate used in neutral culture media has an antimicrobial effect on the growth ofS.aureus,and other early studies have also reported the antimicrobial activity of sodium lactate added into meat products.Brewer et al.[47]reported that 2% or 3% sodium lactate added to fresh pork sausage delayed microbial deterioration,pH decline,and development of sour and off- flavors by 7–10days at 4°C and appeared to protect the red color of products.Lamkey et al.[48]reported that 3%(w/w)sodium lactate was effective in maintaining low microbial numbers,and the shelf-life was extended by more than 2 weeks when fresh pork sausage was stored at 4°C.The results of the present study showed that 48g/kg(i.e.,4.8% w/w)sodium lactate mixed into cooked pork sausage has a significant inhibitory effect on the growth ofS.aureusand SEA production.It was also observed that the antimicrobial effect of sodium lactate depended on its concentration,i.e.,the inhibitory effect became valid only when the sodium lactate concentration in the sausage sample was increased to 24g/kg.

5.Conclusion

Although the Chinese National Standard GB2760-2014 recommends the use of sodium nitrite,nisin,potassium sorbate,and sodium lactate as preservatives in cooked meat products,the results of the present study indicated that their inhibitory effects onS.aureusgrowth and SEA production in cooked pork sausages were quite different.Sodium nitrite,nisin,and potassium sorbate had a weak effect againstS.aureusgrowth and had no effect against SEA production,but sodium lactate could significantly inhibitS.aureusgrowth and SEA production.The results also showed that the antimicrobial effect of sodium lactate depended on its concentration,wherein a concentration＜12g/kg showed no inhibitory effect,but when the concentration was increased to 24g/kg,sodium lactate could effectively inhibit the growth ofS.aureusand SEA production,and the concentration of 48g/kg showed a significant inhibitory effect.Considering the cost and effects,24–48g/kg of sodium lactate is probably the most appropriate concentration for the formulation of cooked pork sausages.

In the present study,the effects of the preservatives when used alone were investigated.Today,combined formulations of preservatives are being suggested,which would exhibit an enhanced antimicrobial effect.This research will be carried out in our future study.

Acknowledgments

This study was supported by the following projects:

Development of Application Technology Project(No:2015-114)issued by Science and Technology Committee of Shanghai Municipal Government.

National Key Scientific Instruments Project (No:2013YQ150557)issued by Ministry of Science and Technology of the P.R.China.