The efficiency of Alginic acid, Sodium alginate and Egg white lysozyme in sustain the safety of beef burger

Abstract Alginates and its salts specially sodium and potassium and its oligosaccharides in addition to egg white lysozyme have attracted many interests in applied researches due to their positive impact on consumer health through potential antifungal and antibacterial, antioxidant, probiotic, antihypertensive, antidiabetic, antitumor, anticoagulant properties and many other benefits. The evidence is that it was used as a probiotic, feed supplements for aquaculture and poultry as well as cryoprotector for frozen foods. Hence, in this study, we tried to use alginic acid (AA), sodium alginate (SA) and egg white lysozyme (EWL) with different concentrations, either individually or collectively in beef burger. The obtained results showed that 2% AA was the best concentration that caused the most pronounced significant reduction (P<0.05) of Staph. aureus count (from 4.93 log10±0.01 as control to 4.14 log10±0.06 as treated) with reduction rate of 0.79 log10 (16.02%) as compared with 0.5 & 1% AA. In contrary, 2% AA showed weak reduction activity of E. coli count (from 4.95 log10±0.01 as control to 4.48±0.04 as treated) with 0.47 reduction rate (9.5%). The same effect was recorded for SA as 2% had the most pronounced reduction for Staph. aureus count which recorded 4.30 log10±0.04 with reduction rate and incidence of 0.63 (12.78%), while for E. coli, it was recorded 4.78 log10±0.01 with 0.17 log10 reduction rate (3.43%). By the same way, it was found that 200 ppm EWL was more effective in controlling of both Staph. aureus (from 4.93±0.01 to 3.98±0.03) with 0.95 reduction rate (19.27%) and E. coli from (4.95±0.01 to 4.45±0.02) with 0.5 log10 reduction rate (10.1%). The obtained results proved that a mixture of 200 ppm EWL + 2% SA was most effective concentration among all other treatments used in the present study which recorded 1.04 log10 reduction rate for Staph. aureus count (21.1%) and 0.75 (15.15%) for E. coli. and recommended to be used in food products as antimicrobial combination in competing both organisms used through the present study. Keywords: Staph. aureus, E. coli, Alginic acid (AA), Sodium alginate (SA), Egg white lysozyme (EWL), Natural antimicrobial activity, sustain safety product. beef burger. Introduction Microbial food spoilage is responsible for deterioration of food as reduction in the sensory attributes, nutritional quality and subsequently, great economic losses. Furthermore, wide spread of the food-borne pathogens leads to food poisoning and damage to the consumer health and loss of food safety parameters. Multiple trends nowadays are encouraged to replacement of synthetic additives and use of new natural antioxidant and antibacterial substances with a possible role as nutritional agents. Multiple functionality seaweed extracts could be incorporated into foods as natural preservatives to enhance the food quality, safety and stability (Vijayavel and Martinez, 2010; Gupta and Abu-Ghannam, 2011 and Cox et al., 2014). Consumers trust and prioritize to food products that have transparency in its contents ingredients/additives that are natural, with reliable names and are appropriate to be good for consumer health (Brewer, 2011). In recent years, 2 Animal Health Research Journal Vol. 8, No. 2, June 2020 Tolba et al. there also have been concerns about food safety due to an increasing occurrence of foodborne illness outbreaks caused by pathogenic microorganisms (Tajkarimi et al., 2010). In order to satisfy consumer’s demands and retrieve its confidence in the safety of food products, those in charge of the food industry should search for natural alternatives to food additives that have strong antioxidant and/or antimicrobial properties. (Fernandez-Lopez et al., 2005 and Ahmad et al., 2015). Algins/alginates are available in both acid and salt forms, alginate is a natural anionic polysaccharide extracted from seaweed, which is composed of β-(1–4) linked D-mannuronic acid and α-L-guluronic acid units (Zheng and Kohn, 2014 and Aziz and Karboune, 2018). Salts of alginic acid including Na+ , K+ , Mg2+ and Ca2+out of which, sodium and calcium ions are considered the most effective cations and are commonly used as the gelling agents and they are extracted from brown seaweeds cell walls and also of the intracellular matrix of the brown algae (Phaephyceae) mainly, Laminaria hyperborean, Macrocystis pyrifera, Ascophyllum nodosum; lesser extent Laminaria digitate, Laminaria japonica, Eclonia maxima, Lesonia negrescens, Sargassum spp.(Skurtys et al., 2010; Kraan, 2012; Hay et al., 2013 and Vera et al., 2013) and some bacteria including Azotobacter vinelandii and pseudomonas (Thomas et al., 2013). Furthermore, Alginate succeeded to be produced from Marine algae (Peteiro, 2018). The molecular weight of alginate ranges generally between 500 and 1000 kDa. (Cha and Chinnan, 2004 and Usov, 2013). In some researches, the term "algin" is used instead of alginate. The goal of the extraction process of sodium alginate is to obtain a product in a dry powdered form. The calcium and magnesium salts do not dissolve in water, while sodium salt is able to be dissolved in water and has a unique swelling, gelling, and mucoadhesive properties (Cardoso et al., 2016 and Szkalska et al., 2016). Alginate Oligosaccharides (OLG) has been reported that they possess antioxidant, anti-inflammatory, and antibacterial properties (Han et al., 2019). Seaweed produces metabolites aiding in the protection against different environmental stresses. These compounds showed antiviral, antiprotozoal, antifungal, and antibacterial properties which aids in control of new diseases or multi-resistant strains of pathogenic microorganisms (Perez et al., 2016). Alginic acid (AA) and sodium alginate (SA) are widely used agents because of their high antimicrobial efficacy and cost effective. Their antimicrobial effects are based on the increase in proton concentration thereby, lowering the external pH. Furthermore, they may affect the integrity of microbial cell membrane or cell macromolecules or interfere with nutrient transport and energy metabolism, causing bactericidal effect (Ricke, 2003). Mixtures of alginate with organic acids or essential oils could exert a wider antimicrobial activity (Theron et al., 2010). New approaches with natural antimicrobial features, which characterized by i) its more potent ii) less hazardous to the consumers health iii) prolonged action are of very interest nowadays. Consequently, antimicrobial that based on natural origin, such as alginates and its salts which could be obtained from various agro-industrial sources are being studied increasingly. (Andrade et al., 2004 and Kakita and kamishima, 2008). Many food products are perishable by nature as well as by the action of bacteria contaminating food during food production, preparation, processing, storage, distribution and handling which considered nowadays the major challenges facing food industry due to its effect on both food safety and quality, some of these microorganisms such as Escherichia coli, Staphylococcus aureus, Salmonella, Listeria monocytogenes and many other organisms that can potentially cause food-borne illness (LòpezMalo et al., 2005). Lysozyme is a natural enzyme obtained from egg white which have a wide spectrum of antimicrobial activity against food-borne pathogens and spoilage bacteria (Gutierrez et al., 2008 and 2009). Lysozyme as a food preservative inhibits the growth of deleterious organisms, thus improve 3 Animal Health Research Journal Vol. 8, No. 2, June 2020 pp. 1-14 the product safety and prolonging its shelf life. Lysozyme also has been used to preserve seafoods, meats, sausages, different kinds of cheese and fresh fruits (Proctor and Cunningham, 1988). Lysozyme is one of the important proteins found in egg white which represent about 3.5% of total egg white proteins (Ibrahim et al., 2007). There are many forms of lysozyme found in nature, but the one found in egg is considered as the most soluble and stable (Benkerroum, 2008). Enzyme can hydrolyze the β-linkage between N-acetylneuraminic acid and N-acetylglucosamine in bacterial cell walls. Along with the antioxidant substances, the antimicrobial properties of different preservatives are required to fulfil the quality and safety parameters that compliant with consumer demands, satisfaction and their confidentiality, these substances or products including organic acids, alginic acid and its salts, egg white lysozyme, essential oils, herbal products and phenolic compounds (Basuny et al., 2012). The present study was conducted to determine the efficiency of the antimicrobial activities of alginic acid (AA), sodium alginate (SA), egg white lysozyme (EWL) and a combination of EWL with SA by using different concentrations against Staph. aureus and E. coli experimentally inoculated in beef-burger. Materials and Methods preservatives used in the established experiment: Egg white lysozyme (EWL) AR, BIO BASIC CANADA INC., CAS:12650-88- 3.Alginic acid (AA) AR, AVI-CHEM LAB., MUMBAI-INDIA, CAS: 9005-32-7. Sodium alginate (SA) AR, AVI-CHEM LAB., MUMBAI-INDIA, CAS: 9005-38-3 All preservatives which have been used in this study were of analytical grade and watersoluble ingredients and the doses of the preservatives used in the present study (0.5, 1 and 2% of both alginic acid and sodium alginate as well as 100, 150 and 200 ppm of egg white lysozyme) were recommended by several investigators who have used the same or even more than the concentrations in the present study as they suitable to preserve quality characteristics and does not alter the sensory attributes (firmness, color and odor) of food product (Corradini and Innocente, 2002; Walewijk et al., 2008; and Gammariello et al., 2009). Sample preparation: One beef meat sample weighted around 4500 g was purchased from butcher shop in Cairo to perform one experiment, where the experiment was repeated three successive times to obtain the mean ± SD, the bulk sample was transferred under strict hygienic measures to laboratory as soon as possible, minced with addition of ingredients required for production of beefburger. Manufactured beef-burger was divided into five groups, each group contained six samples (a total of 30 samples of 150 g each); 15 samples were contaminated with 5 log10cfu/g Staph. aureus and the other 15 samples were contaminated with 5log10cfu/g E. coli and treated as follows: The 1st group; three contaminated samples with Staph. Aureus and three contaminated samples with E. coli were kept as control positive to estimate the initial bacterial load of both organisms. The 2nd group; three contaminated samples with Staph. Aureus and treated with 0.5, 1 and 2% of AA, separately and the other three contaminated samples with E. coliwere treated with 0.5, 1 and 2% of AA. The 3rd group contained six contaminated samples as mentioned in the 2nd group and treated with 0.5, 1 and 2% SA. The 4th group contained six contaminated samples as mentioned in the 2nd group but treated with 100, 150 and 200 ppm EWL. The 5th group contained six contaminated samples as in 2nd group but treated separately with a mixture of SA and EWL (0.5% & 100 ppm), (1% and 150 ppm) and (2% & 200 ppm). The experiment was repeated three times to carry out the statistical operations. 4 Animal Health Research Journal Vol. 8, No. 2, June 2020 Tolba et al. Preparation of tested strains Working solution of Staph. aureus and E. coli were prepared from reference stock solution stored at -80˚C in cryovials. One bead was resuspended in brain heart infusion broth (Oxoid) and incubated overnight at 37˚C for 24 hour prior to the experiments to obtain a final viable count of about 109 CFU/ml, serial dilution was made using physiological saline to obtain approximately 105 CFU/ml which used to contaminate the ground beef used in the manufactured of. beef burger, while conducting the experiment under complete aseptic condition. Preparation of the samples and serial dilution (APHA, 2001): Twenty-five grams of each sample was transferred aseptically into stomacher bag and stomached with 225 ml of 0.1% sterile peptone water. Transfer by means of pipette 1 ml of the initial suspension into a tube containing 9 ml of sterile diluent. Mix thoroughly by using vortex for 5-10 seconds to obtain 1:100 dilution. Repeat this operation to obtain dilutions 1:1000, 1: 10000 and etc. dilutions. Enumeration of Staphylococus aureus (FDA, 2001) About one ml. of food homogenate was transferred and distributed over the surface of 3 plates of Baired-Parker agar (eg. 0.4 ml, 0.3 and 0.3 ml), using sterile bended glass spreader. The plates were retained in upright position until inoculum is absorbed by agar for about 10 mints, or placed in upright position in the incubator for about 1 hour. The plates were inverted and incubated for 24-48 hours at 35oC and examined for determination of Staph. aureus count. Enumeration of β-glucuronidase - positive Escherichia coli according to (ISO 16649- 2:2001) (TBX method): This method for enumeration and isolation of β -glucuronidase–positive Escherichia coli in all kinds of food and feed of animal origin, by growing the organism on tryptone –bileglucuronide medium (tbx) at 44oC for 24 h. Positive plates showed blue green colonies. Statistical analysis: - Statistical analysis of the obtained data was run in triplicate by using of Statistical Packaging for the Social Science (SPSS) Ver. 20.and the results were expressed as mean and standard deviation (Mean±SD). Data were analyzed using analysis of variance (one-way ANOVA). The results with p-value less than 0.05 (p ≤ 0.05) was considered statistically significant. List of preservatives versus the concentration of each substance were listed in Table (A) Table (A). Type and concentration of different preservatives used in the current experiment Preservatives used Concentration Alginic acid (AA) 0.5 % Alginic acid & Sodium alginate (AA) 1 % Alginic acid & Sodium alginate (AA) 2 % Sodium alginate (SA) 0.5 % Sodium alginate (SA) 1 % Sodium alginate (SA) 2 % Egg white lysozyme (EWL) 100 ppm Egg white lysozyme (EWL) 150 ppm Egg white lysozyme (EWL) 200 ppm Combination of both SA + EWL 0.5 % + 100 ppm Combination of both SA + EWL 1 % + 150 ppm Combination of both SA + EWL 2 % + 200 ppm 5 Animal Health Research Journal Vol. 8, No. 2, June 2020 pp. 1-14 Results and Discussion Most food products required strict protection against food poisoning and food spoilage bacteria which gained access to the food as a result of contamination during processing and storage operations. Also, consumers demand for safe product with natural preservatives if required, which promoting food producers and researchers to look for alternatives in order to get safe products and good storage practices which will result in improving product quality and mitigate microbial risk levels without causing nutritional losses and organoleptic changes. In this context natural preservatives are gaining a great interest from research and industry, due to the potential to provide quality and safety benefits, with a reduced consumer health hazard (Lucera et al., 2012). It was observed from the data recorded in Table and Fig. (1) that the most pronounced reduction rate for Staph. aureus was in the samples of the 2ndgroup which treated with 2% of AA as compared with the other two concentrations (0.5, 1%) which used for treatment of the other samples in the same group, as the count was reduced to (4.14 log10±0.06) with reduction rate of 0.79 log10 (16.02%) as compared with the 1stcontrol group (4.93 log10±0.01), followed by 1% AA which recorded Staph. aureus count of 4.60±0.02with reduction rate represented by 0.35 log10 (7.1%). On the other side, SA with 2% concentration represented for the 3 rd group was also the most effective one in reducing Staph. aureus count to 4.30 log10±0.04 with reduction rate of 0.63 log10 (12.78%), followed by SA 1% who had achieved reduction rate of 0.15 log10 (3.04%). From the obtained results, it could be concluded that Staph. aureus counts were reduced significantly (P<0.05) in all treatments except, there was no significance difference (p>0.05) between control samples and that treated with 0.5% SA. Despite these recorded results, there are only two concentrations assimilated by 2% of both AA and SA which had reduction rate exceeded more than 0.5 log10 (0.79 and 0.63 log10cfu/g) respectively, which cleared that 0.5% SA found to have the least antimicrobial activity against Staph. aureus among the other concentrations of AA of the 2nd group and SA of the 3rd group. Table (1) also showed that E. coli was less affected by either AA or SA as compared with Staph. aureus, in which 2% concentration of AA resulted in reduction of E. coli by 9.5% (0.47 log10), followed by 1% (0.23 log10 / 4.67%). While 2% SA was recorded reduction rate of 0.17 log10 (3.43 %) followed by 1% (0.11 log10 / 2.22%).Statistical analytical results showed that there were an obvious significance differences (P<0.05) of E. coli counts between all samples, excluding, the difference was not significant (p>0.05) between treated samples with 0.5% SA and each of control samples and samples treated with 0.5% AA. Although, there were a significant differences between most of the different treatment concentrations of both AA and SA and control samples for E. coli count, the anti E. coli effect of all treatments as the microbial reduction rate has no tangible effect which does not exceed 0.5 log10 which is considered in all measures as a weak or low effect. It was generally obvious from results in Table and Fig (1) that; (i) The higher the concentration, the more the antimicrobial activity (2% was the best concentration followed by 1% and finally, 0.5% which considered weak and nonsignificant). (ii) All tested concentrations of AA and SA had a marked reduction effect on Staph. aureus as compared with its very weak effect on E. coli (iii) Since the reduction rates of both AA and SA on either Staph. aureus as an example of Gram-positive bacteria or Gram -negative E. coli were not significant, it is not recommended to use either AA or SA alone as antimicrobials but it is preferred to be used in combination with another synergistic antimicrobial substances.This agreed with Scott and Strong (1964) as they indicated that the value of sodium alginate in controlling Staph. aureus food-poisoning microorganism in frozen food is questionable. Also, the obtained results were compatible with Mhadhebi et al. (2012) who found the extract of 24 screened organic fractions of 6 seaweeds from the Tunisian Mediterranean coast were exhibited moderate to weak activity against Staph. aureus; Staph. epidermis, E. coli and Micrococcus luteus. This also 6 Animal Health Research Journal Vol. 8, No. 2, June 2020 Tolba et al. substantiates the findings of Kim et al. (2000) and Hay et al. (2013) who concluded that Alginic acid and its salts may have other effective functions more important to food than being antibacterial as thickening agent, stabilizer, emulsifier, chelating agent, encapsulation, swelling, a suspending agent, or used to form gels, films and membrane. Table (1). Mean count and reduction rate of Staph. aureus and E. coli using Alginic acid and sodium alginate. Antimicrobials Bacterial counts and reduction values (Log10cfu/g) Group Concentration Staph. aureus E. coli Mean±SD Reduction Mean±SD Reduction Rate % Rate % Control 0.0 4.93a ±0.01 --------- -------- 4.95a ±0.01 ------- ------- AA 0.5 % 4.84b ±0.02 0.09 1.83 4.90b ±0.01 0.05 1.01 1.0 % 4.60c ±0.02 0.35 7.10 4.70c ±0.03 0.23 4.67 2.0 % 4.14d ±0.06 0.79 16.02 4.48d ±0.04 0.47 9.50 SA 0.5 % 4.90a ±0.02 0.03 1.55 4.93ab±0.01 0.02 0.40 1.0 % 4.78e ±0.01 0.15 3.04 4.84e ±0.01 0.11 2.22 2.0 % 4.30f ±0.04 0.63 12.78 4.78f ±0.01 0.17 3.43 Mean ± standard deviation (n=3); Means in the same column with different superscripted letters are significantly different (p<0.05). Fig. (1): Reduction rate (log10cfu/g) of AA and SA on Staph. aureus and E. coli 7 Animal Health Research Journal Vol. 8, No. 2, June 2020 pp. 1-14 Moreover, many investigators (Nair et al., 2005; Aguila-Ramírez et al., 2012; Nogueira et al., 2014; El Wahidi et al., 2014and Karthikeyan et al., 2015) found that methanolic extract of AA and SA from seaweeds (L. johnstonii, D. flabellata and U. lactuca) from the Gulf of California showed activity against Staph. aureus, while it poses no observed activity against E. coli. These results were compliant with that obtained in the present study as the AA and SA found to have a little or low antimicrobial activity against E. coli in comparison with their slightly to moderate effects on Staph. aureus. While, Osman et al. (2010) and Dhanya et al. (2016)observed the antimicrobial activities of crude extracts from the species of Rhodophyta, Chlorophyta, Ulva reticulata and Phaeophyta against bothof Staph. aureus and E. coli. Such differences in results may be attributed to the difference of in types of seaweeds used and the seasonal variation of antimicrobial activity of the extract. (Padmakumar and Ayyakkannu, 1997). Moreover, Karbassi et al. (2014) attributed the presence of antimicrobial activity of AA on Staph. aureus more than E. coli due to the difference in the cell wall structure and physicochemical characteristics of both microorganisms, in which Staph. aureus have an outer cytoplasmic membrane made from lipopolysaccharide which is easily adhered and penetrated by AA, while E. coli contained rigid peptidoglycan layer outside the cytoplasmic membrane which in unlikely to be penetrated by AA The U.S. Food and Drug Administration (USFDA, 2018) had classified food grade sodium alginate as GRAS (generally regarded as safe) substance in Title 21 of the Code for Federal Regulations (CFR) and listed its usage as an emulsifier, stabilizer, thickener and gelling agent. Furthermore, the European Commission (EC) listed alginic acid and its salts (E400– E404) as an authorized food additive (Younes et al., 2017). Alginate is widely used in various industries such as food, beverage, textile, printing, and pharmaceutical (Kim et al., 2000 and Hay et al., 2013). Sodium alginate is the most common salt of alginic acid (Yoo and Krochta, 2011).In this regard, European Food Safety Authority (EFSA, 2017) mentioned that Alginic acid and its salts (E 400–E 404) are authorized to be used in a