Experiment on Cheese Spoilage: Fungal Growth and Mycotoxin Migration

Abstract

Despite the food packaging was employed as part of food preservation concept. Exposed surfaces of cheese are vulnerable to mold contamination from several sources. Thus, cheese spoilage is generally confined to molds. Four mycotoxigenic fungal isolates were used in this study; new method was applied in this study as a safe alternative cover to conventional covers in certain semi hard cheeses which are based on chitosan/potato starch and / or thyme, safflower and sesame powders. These new covers were adjusted for thickness homogeneity, covers permeability to water vapor was tested using the standard methods of ASTM, with certain modification .

Other new cover properties were adjusted.

This study aimed to investigate the effects of these covers on fungal growth and Mycotoxins migration to medium which mimicked cheese structure. Our results illustrated that The enriched chitosan covers with (HPPs) proved to be an efficient method to increase the antifungal activity and to reduce the inherently high water vapor permeability of plasticized chitosan packaging covers .

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The use of chitosan cover alone or enriched with HPPs succeeded also to control the growth of the tested fungi invading cheese and reduced the mycotoxins migrated amounts cheese which . The response of EVOH with and without herbal plant parts powders (Hppps) addition was also discussed, the addition of these plant material ameliorated the capability of EVOH to reduce fungal growth and the majority of the tested mycotoxins migration amounts. Our results may be realized a significant impact on shelf-life extension and cheese safety during transport and trade.

Introduction

Cheese is considered a very important food. Semi hard cheese like Edam and Gouda cheese are among the very popular cheese around the world. (Official Edam Town website, 2007, Edam com., 2007, European commission, 2010, Miller et al., 2013and Marriam Webster, 2015). Gouda cheese is very susceptible to mold growth and is normally kept under refrigeration. Both of two semi hard cheese was surrounded by an interior coat or packs which permitted gas exchange. However, molds are ubiquitous in cheese. Mold mycelia and mold spores are dispersed by air currents in the cheese plants. Molds also grow on damp surfaces such as walls and shelves in cheese curing rooms and in storage room too. Exposed surfaces of cheese are vulnerable to mold contamination from these sources.

Thus, cheese spoilage is generally confined to molds, which are psychro tolerant and can grow under conditions of relatively Low oxygen. The low aw requirements of molds allow them to grow at relatively high salt concentrations. Those properties allow molds to grow well on Cheese surface. Certain researchers were studied hard, semi hard and semi soft cheeses from Denmark, France, Greece, and U K. they found that the main fungal isolates were Penicillium species such as P.citrinum and P. verrucosum followed by Aspergillus species.. Mycotoxins are secondary metabolites of molds that have adverse effects on humans that result in illnesses and economic losses. Aflatoxins ,ochratoxins are mycotoxins of greatest importance (Kure et al., 2001 and 2004;EFSA,2012 and Doughari,2015).

Petrochemical-based polymers predominate in food packaging due to their easy processing, excellent barrier properties, low cost and the need to use plasticizing agents to obtain stretchable ?lms (Kowalczyk and Baraniak, 2011; and Tapia, 2013). The composition of Flexible multilayer film packaging cheese consists of EVOH (Ethylene-vinyl alcohol) copolymer which is used in coextruded plastic film to improve oxygen barrier properties (Bendall,2007, Gordon ,2009). Embodiments of the present invention for using a core layer having a greater amount of EVOH ( a saponified or hydrolyzed copolymer of ethylene and vinyl acetate 70-80%) and lesser amounts of nylon (20-30%) to produce a film having a low CO2 gas transmitting rate, particularly when using an EVOH copolymer having an ethylene content of about 48 mole percent. which are suitable for packaging articles which respire low levels of CO2 such as gouda, , edam, butter kase and cheddar cheeses, by blending lower amounts of EVOH with higher amounts of nylon.(Edwards et al.,2003).

Certain reports (Levitt,1998, Goulas et al., 2000, Goulas et al., 2007, Bendall et al.,2007, Cruz et al.,2008 and Kanishka et al.,2013) illustrated the migration of undesirable substances to cheese like phtallate, dioxin and diethyl hexyl adipate (DEHA) plasticizer , ink and hormone-mimicking chemicals, from interior coats into hard and soft cheese. But the migration of mycotoxins throw these packs are not studied enough. However, simple changes in packaging materials are required to solve all these problems and to avoid aggravating this draw back. Environmental concerns enhance and stimulate the use of renewable and safe resources for producing economically convenient applications that could also improve life quality (Garca? et al.2004). Chitin is the natural polysaccharide biologically produced by living creatures on the earth in huge quantities. It is estimated that total production of Chitin on the earth annually is about 1 to 100 billion ton which is available for commercial use annually.

However, only a few thousand tons are actually used in the world. Chitosan, the N-deacetylated form of chitin, is a natural cationic polysaccharide, which is edible, non-toxic, biodegradable, biocompatible (No et al., 2007) and commercially available, that has been employed in a variety of applications (Muzzarelli, 2010&Muzzarelli et al., 2012). Moreover, chitosan is also well-known for its broad antimicrobial activity against bacteria and fungi (Muzzarelli et al. 1990, Cagri et al. 2004; C?rdenas et al.,2008). Chitosan ?lms have a low permeability to gases (CO2 and O2) and good mechanical properties. (Ojagh et al., 2010). Its safety has been verified scientifically through many tests. This food waste transformation not only reduces the waste from industries, it also reduces the cost of raw materials (Siracusa et al., 2008 and Janjarasskul et al,.2010). Essential oils (EOs) have been long recognized for their antibacterial, antifungal, antiviral, insecticidal and antioxidant properties.

They are widely used in medicine and the food industry for these purposes. (Henri et al.,2012 and Zareef et al.,2018). It has been reported that EOs containing aldehydes or phenols, such as cinnamaldehyde, citral, carvacrol, eugenol or thymol as major components showed the highest antibacterial activity, followed by EOs containing terpene alcohols. Other EOs, containing ketones or esters, such as ?-myrcetene, -thujone or geranyl acetate had much weaker activity. (Dorman and Deans.2000; oussalah et al.,2007; Dicko.2010 ; Aiouazzou, 2011and Bopitiya and Madhujith, (2013). Phenolic acids are a major class of phenolic compounds, widely occurring in the plant kingdom (Cai etal.,2004). predominant phenolic acids include hydroxybenzoic acids (e.g., gallic acid, p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, and syringic acid) and hydroxycinnamic acids (e.g., ferulic acid, caffeic acid, p-coumaric acid, chlorogenic acid, and sinapic acid) (Cai et al., 2006).

Natural phenolic acids, either occurring in the free or conjugated forms, usually appear as esters or amides (Karimkhan et al., 2016) Sesame seeds (seasamum indicum), Safflower petals (Carthamus tinctorius L and and thyme leaves (Thymus vulgarisL ) are containing phenolic acid ,oils such as oleic and linoleic, palmitic acid and stearic acid in sesame Sesamum indicum (Hall, 2003; Zengin and Ayse, .2014). It is also rich in various bioactive compounds and phytochemicals (Cho et al.,,2000 Zuo etal.,2013,Chen et al.,,2013and Furumoto etal.,2016) which are known to play an important role in providing stability against oxidation of oil and contribute to antioxidative activity (Shahidi et al., 1997; Philip et al., 2010;Wu et al.,2013and Nidhal etal.,2014). Currently mold control in cheese generally involves the use of approved chemical preservatives (generally recognized as safe, GRAS). These special packaging techniques add to the cost of production, which is passed onto consumers.( Magnusson et al., 2003 and DanZhao, 2011 ).

So, to solve this problem further cheap and safe package structures are needed ,therefore, the objective of this work was to evaluate the combined use of chitosan, potato starch and /or herbal plant powder (hpp) as enriched chitosan potato starch cover surrounding semi hard cheeses to reduce the water vapor permeability of chitosan ?lms intended as edible food cover packaging improving at the same time, their antimicrobial to reduce the food spoilage rate and the migration of mycotoxins to cheese. Moreover, this antimicrobial covers may be improve the concept of active packaging which can be reduce , or delay the growth of microorganisms on the surface of food in contact with the package and consequently inhibit the mycotoxin transition to packaged cheese . This work aimed also to study the performance of biodegradable and edible biopolymers and their combination with (hpp) as natural packages for selected food products.

Matherials and Methods

1. Fungal strains: Strains of Aspergillusflavus, Aspergillus niger, Penicillium citrinum and penicillium verrocosum were brought from Agriculture faculty of Shatby. Plant Pathology Department. Alexandia University. The ability of each isolate to produce mycotoxin was tested using plug agar method according to (Bragulat et al.,2001) then the mycotoxin amount was quantified by HPLC technique according to (Turnera et al., 2009 ). All mycotoxins standards are brought from Sigma Aldrich. Cairo Office. Fungal strains sources are illustrated in table n?=1.
2. Plant powder : Sesame seeds, thyme leaves and safflower (Carthamus) petals are brought purified then mycotoxins occurrence were detected using multi analysis TLC methods according to Rudolf et al.,2008 Only Mycotoxins free plant materials were used.
3. Preparation of plant extract: Sesam seeds, Carthamus petals and thyme leaves are purified, surface sterilized by ethyl alcohol 97% then oven dried at 45?C for 36 hrs. (according to Burt and Reinders. 2003). These plant material ethyl extracts are prepared according to Das et al., 2010 .
4. Detection of phenolic compounds: Plant-derived polyphenols receive considerable interest because of their potential antioxidant and antimicrobial properties. Undoubtedly, it is very important not only to determine those properties but also to determine each and every phenol in aromatic plants both qualitatively and quantitatively. The main phenolic compounds occurred in each tested materials was detected using Mass spectrophometry apparatus according to Charalampos etal.,2008. The resulted data were registered as illustrated in tables (2, 3 and 4 ).
5. Potato starch extraction process: Extracting starch from potatoes was carried out according to Anne, 2005. Grated peeled Potatoes are water rinsed for several times. Then, the grated potatoes are water immerged then put into a sterilized cloth gauze .Squeeze process was repeated until almost all potato starch content was released. Potato starch was taken after centrifugation and the precipitated starch was oven dried at 45?C for72 h.

Testing the role of thickness in mycotoxin migration through different covers

Preparation of the tested covers: Three kinds of cover are used in this experiment:

• The conventional cover: The cheese conventional cover ( multilayer EVOH) was gently removed, cut into disks 9x9cm, washed several times with alcohol 96% and sterilized water to remove any fatty traces then surface sterilized with formalin 1% then washed thrice with sterilized deionized water . The cover thickness was measured using a manual Micrometer apparatus. The cover thickness was registered as 0.7 mm (or 0.07 cm or 700 Micrometers).
• The chitosan cover: The chitosan cover was prepared by adding 15 g. potato starch to , 1g. chitosan, both of sterilized vegetable glycerine (15g.) (Chilo et al.,2008 and Mahdavi et al.,2018) and lactic acid (5g.) are add to 100 ml hot deionized water to save elasticity to the cover (Il'ina andVarlamov.2003) porosity (Luciano et al.,2001) and tensile trenching(Zhong and Xia. 2008 and Pradhan et al.,2017) . The mixture was gently stirred and heated at 65? C for 15 min. Trial cover production was carried out by weighing the mixture amount which added to each Petri dish separately several time, drying the produced covers and measuring their thicknesses in several points around the cover according to (Mahdavi etal.,2018) until we obtained the desired thickness of the cover. After that the desired amount of mixture (0.75 g.) was added to each Petri dish then dishes were oven dried for 2hrs. at 45?c .Each herbal powder(1% Conc. w/w) was gently homogeneously dispersed into chitosan dish before drying process in only fortified chitosan treatments before oven dryness according to Pereda et al.,2010 with certain modification . The obtained chitosan cover physical properties were adjusted using Barbara and Olech tables according to Barbra and Olech,(1996) then was measured using Micrometer apparatus and registered ( 0.7 mm or 0.07 cm or 700 Micrometers).
• The cellophane cover: Cellophane cover (thickness 0.004cm (40 micrometer) was employed as a second low barrier cover. Cellophane thin transparent sheet was cut into disk 9 x9cm then the two side's surface of each disk was sterilized by spraying ethyl alcohol 96% and putted in oven at 45'c for one hour.
• Testing water vapor penetration and diffusion across the covers: This test was carried out to cover the determination of the amount of water vapor passes through covers to substrate which consequently affected their barrier properties . These amounts are quantified by the weight of mass under every tested cover using Hes and Queir's (1996) method ;Zhongbin et al.,2001 Desorption Henry's law concept (Siracusa, 2012) and the standard methods of ASTM, 1996 No. 96 E (Mahdavi etal.,2018)and ASTM D1653-13 (ASTM,2018) with certain modification.

All the used covers were cut similarly to have the same surface. Then, three cells containing silica gel (1g) each were covered with one of the tested cover coated with molten paraffin as a blank [c]. Afterwards, another twelve cells were placed inside the desiccators containing silica gel (1g) and each cell was covered by one tested cover (three cells) but the other cells were covered by one hpp enriched cover . Water at 25°C produces 100% moisture. So, water was poured into the cell permeability measurement. The difference in moisture on the two sides of each cover varied according to the cover structure. The difference in moisture was registered after 24hrs. by weighing the silica gel under each tested cover before [A] and after heating it [B] in oven at 105 ? C for one hour then the resulted water vapor desorbed from each cover and absorbed by silica gel was calculated according to the equations

[A- B]) /A x100

The difference between them was the water vapor permeability (WVP) or desorption amount of WV reached food.

Where B = Weight of the oven dried cell containing silica gel coated with cover

C = Weight of the cover+ molten paraffin film.

C' = Weight of the oven dried cell containing silica gel and covered with one tested cover+ molten paraffin film.

N.B = -The difference between c and c' = zero.

Changes in cell weight are relative to time.

Testing the mycotoxin migration through the three tested covers under cheese storage conditions

This experiment was carried out according to Elad et al.,1983 with certain modification to determine the capability of the conventional cover and the proposed cover to prevent the tested mycotoxins to reached the medium (which represented cheese) in comparison to cellophane (a very thin sheet) to investigate the role of cover thickness in mycotoxin migration during storage process. A modified medium consisting of Dextrose casein peptone-Agar medium (Merk, Germany) (15g) and PDA (125g) added to 1L.distilled water are used in this experiment. Four groups of petri dishes 13 dishes each were filled with the mention medium and then the tested cover was placed upon the solidified medium. Each dish was inoculated by the tested fungus. All dishes groups are stored for six weeks in a cooling incubator at 40?F and RH 68% as recommended by cheese producers.

Effect of the tested covers on fungal growth

At the of the storage period, dishes are taken then the radial growth of each fungus was measured. Data are registered as shown in the table 1.

Table 1: Effect of Cheese Covers on Fungal Growth

Effect of the tested covers on mycotoxin migration to substrate

Each tested cover was gently removed then plugs 4 mm each was taken from the medium of each petri dish (ten plugs from each perti dish). The mycotoxins migrated to the medium through each tested cover was determined individually using plug method agar according to Bragulat et al.,2001 then each mycotoxin was quantitatively detected using HPLC according to Turnera et al., 2009.Data are registered as a illustrated in table 2.

Table 2: Mycotoxin Migration through Cheese Covers

Statistical analysis

The experiment was carried out in three replications in a completely randomized design. The data were subjected to statistical analysis using Costat computer package (CoHort Software, Berkeley, CA, USA). One way ANOVA was used and comparison between the resulted data was done using least significant difference (LSD) according to Duncan's Multiple Range test was applied to compare the treatment mean values according to McDonald, (2009).

Results

Table 3: Mycotoxin Production by Fungal Isolates

Table 4: Phenolic Compounds in Tested Materials

Discussion

The results of the study revealed that the fungal isolates tested in the experiment produced varying levels of aflatoxin and ochratoxin, which are mycotoxins of concern in cheese production (Table 1). Among the isolates, Aspergillus flavus exhibited the highest aflatoxin production, while Aspergillus niger produced the highest levels of ochratoxin.

Phenolic compounds were detected in the tested materials, including sesame seeds, safflower petals, and thyme leaves (Table 2). These phenolic compounds are known for their potential antioxidant and antimicrobial properties, which could contribute to the effectiveness of the proposed cheese covers.

Conclusion

In conclusion, this study investigated the potential of using chitosan, potato starch, and herbal plant powders as alternative cheese covers to mitigate fungal growth and mycotoxin migration in cheese-like conditions. The results suggest that these enriched covers have the potential to reduce fungal growth and mycotoxin migration, which could enhance the safety and shelf life of cheese during storage and transport. Further research and development of these natural and biodegradable covers offer promising solutions to address mold contamination and mycotoxin issues in cheese packaging.