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- NOM-121-SSA1-1994: Especificaciones sanitarias sobre los quesos frescos, madurados y procesados.
- NOM 121 SSA1 1994. Quesos Frescos
- 1. Introduction
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Mariano Escobedo, Col. Natural extracts possess several kinds of antioxidants anthocyanins, betalains, thymol, carvacrol, and resveratrol that have also demonstrated antimicrobial properties. In order to study these properties, extracts from cranberry, blueberry, beetroot, pomegranate, oregano, pitaya, and resveratrol from grapes were obtained.
Growth inhibition tests of mesophilic aerobes, coliforms, and fungi were conducted in films prepared from the extracts in accordance with Mexican Official Norms NOM.
Optical properties such as transparency and opacity, mechanical properties, and pH were also analyzed in these materials. The films with beetroot, cranberry, and blueberry extracts demonstrated the best antimicrobial activity against various bacteria and fungi in comparison with unmodified chitosan—starch film. This study shows that the addition of antioxidants improved the antimicrobial performance of these films. It was also found that antimicrobial properties are inherent to the films.
These polymers combined with the extracts effectively inhibit or reduce microorganism growth from human and environmental contact; therefore, previous sterilization could be unnecessary in comparison with traditional plastics.
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The presence of extracts decreased transmittance percentages at and nm, as well as the transparency values, while increasing their opacity values, providing better UV—VIS light barrier properties.
Despite diminished glass transition temperatures T g , the values obtained are still adequate for food packaging applications. In recent decades, human health has been increasingly threatened by microbiological contamination; this can be seen in the spread of food-borne infections and communicable diseases that has occurred recently.
Therefore, it is necessary to find, develop, and improve antimicrobial natural materials capable of inhibiting the proliferation of various microorganisms. Several research studies have shown that chitosan and other several natural antioxidants are natural biocides that are promising candidates for solving this problem [ 1 ]. Chitosan is obtained by the N -deacetylation of chitin, which is the second most abundant natural polysaccharide in nature.
Due to its good biocompatibility, it is widely applied in the pharmaceutical, agricultural, food, nutritional, and biomedical industries, in addition to other applications such as sewage treatment. In recent years, the bactericidal and fungicidal features of chitosan have drawn wide attention [ 2 ].
Chitosan is not soluble in pure water or organic solvents, but it is soluble in aqueous solutions of organic or mineral acids. Chitosan-based materials can be used as degradable and edible films or coatings.
Starch has been used to produce biodegradable films, partially or totally substituting plastic polymers, because of its low cost and renewability. However, the broad applications of pure starch films are limited by the water solubility and fragility of the material [ 3 ]. There are various antioxidant substances found naturally in foods that slow the oxidation reactions in cells from which harmful free radicals originate.
Therefore, antioxidants play a key role in reducing cardiovascular disease, tumors, and neurodegenerative diseases. Furthermore, they strengthen the immune system. These substances have been studied in recent years because of their biocidal and antiseptic properties, as well as their abundance in nature [ 4 ].
Natural antioxidants such as anthocyanins, betalains, thymol, carvacrol, and resveratrol are known for their excellent antimicrobial properties.
Anthocyanins are water-soluble pigments with glycoside features that are widely distributed in the plant kingdom. They are responsible for the reddish, orange, bluish, and purplish coloration of grapes, apples, roses, strawberries, and many plant products, mainly fruits and flowers [ 5 ].
Betalains are natural water-soluble pigments with nitrogen in their structure; they are synthesized from the amino acid tyrosine. Betalains are divided into two groups: betacyanins, which produce a red color, and betaxanthins, which provide a yellow color [ 6 ].
Thymol is a colorless, crystalline substance with a characteristic odor that is present in the essential oils of thyme or oregano. Thymol belongs to the group of terpenes and it has disinfectant and fungicidal features. Carvacrol is an isomer of thymol [ 7 ]. Resveratrol is an antioxidant produced by more than 70 species of plants in response to stressful situations ultraviolet radiation, fungal infections, etc.
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It is present in many foods, such as grapes [ 8 ]. Also, their presence indicates the existence of conditions that can favor the growth of pathogens such as Clostridium botulinium , Cl.
Perfringers , Bacillus mesentericus , B.
High counts of these bacteria indicate that food will soon spoil because of these microorganisms [ 9 ]. The presence of total coliforms is an indicator of potential fecal human contamination and the presence of enteric pathogens [ 10 ].
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Penicillum notatum is a fungus that grows quickly, between 3 and 7 days. It rarely causes fungal disease in compromised patients [ 11 ]. Aspergillus fumigatus can cause allergic aspergillosis, which causes granulomas in the lungs. Rarely does the organism cause diseases in humans [ 12 ].
Aspergillus niger has various industrial applications. It can rarely cause external otitis and allergic aspergillosis [ 13 ]. The innovative purpose of this study is to intensify the biocidal properties of chitosan—starch films against food-contaminant-indicator microorganisms mentioned here by using organic and nontoxic materials, such as natural extracts, with demonstrated antimicrobial properties that are compatible with the matrix film.
By using simple and cheaply obtained extracts from renewable resources, this innovation may also reduce costs. These new biomaterials are promising candidates for food packaging because of their lower transparency, higher opacity, flexibility, and adequate operating temperature. The antimicrobial activity of each film was observed using two series of three tests. A film was considered biocidal if it prevented the growth of microorganisms not only in its surface but also in its periphery.
The antimicrobial activity of these films cannot be explained perfectly, but some researchers have proposed several mechanisms.
The antimicrobial activity of chitosan—starch with natural antioxidants from natural extracts may be attributable to the following mechanisms.
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The electrostatic interaction between positively charged chitosan and some bacteria with negatively charged cellular membranes such as certain types of aerobic mesophilic and coliform bacteria significantly alters the barrier properties of these membranes. The phospholipids present in the cellular membranes of Gram-negative bacteria interact with the NH- groups of the chitosan, causing the bacteria to lose cellular material. The chelating capacity of the chitosan can affect microbial growth.
The use of acetic acid as a chitosan solvent can improve the antimicrobial activity of chitosan according to a study that showed that organic acids with low carbon numbers produce better antibacterial solutions [ 14 ].
The external membranes of bacteria protect them against polar organic compounds, which are harmful to them [ 15 ]. Consequently, it is possible that cell membranes are broken by the action of chitosan, promoting the entry of natural antioxidants into the cells. The antioxidants have antimicrobial properties and can therefore increase the antibacterial properties of chitosan. The interaction between positively charged amino groups of chitosan positive charge is caused by the acid solvent and negative residues in the fungi walls changes the permeability of the fungal plasmatic membrane, altering its main functions.
The presence of chitosan inhibits some enzymatic synthesis on fungi and can favor the occurrence of cytological alterations [ 16 ]. Carvacrol is capable of breaking the external membrane of Gram-negative bacteria, causing the exit of lipopolysaccharides and increasing the permeability of the cytoplasmic membrane [ 17 ].
The presence of carvacrol stimulates the removal of adenosine triphosphate ATP from the cell, inhibiting enzymes and decreasing the proton motive force [ 7 , 18 ]. Thymol is capable of disintegrating the external membrane of Gram-negative bacteria, resulting in the elimination of lipopolysaccharides and increased cytoplasmic membrane permeability. Thymol changes the membrane permeability and permits the loss of essential compounds such as ions, ATP, nucleic acids, and amino acids [ 7 ].
In our tests, most of the films with extracts demonstrated effectiveness against the presence of microorganisms, which is considered the main cause of food decomposition.
After incubation of the microorganisms for a proper period, the number of colony-forming units CFU developed in each case was counted. The limits specified in the relevant standards were considered, and we found that very few films presented numerous colonies. The results and film nomenclature are presented in Table 1. The CFU numbers presented in Table 1 were counted visually.
NOM-121-SSA1-1994: Especificaciones sanitarias sobre los quesos frescos, madurados y procesados.
Films were considered contaminated if they presented a CFU number equal to, or higher than, the number considered in the section 10 of each applicable norm. In the case of mesophilic aerobic bacteria, we found that most of the films with the exception of QSG2, QSG5, and QSO5 in one of two tests inhibited or reduced the growth of mesophilic aerobic bacteria.
In the case of coliforms, it was observed that most of the films with the exception of QSAm0. In the case of fungi, it was observed that most of the films presented better inhibition of environmental fungi than the control chitosan—starch film QS2 with the exception of QSA5, QSB0. In summary, the presence of natural extracts provided the films with a better resistance to environmental microorganisms and human contact, which implies that most of them, unlike traditional biocide polymers, will not require a previous sterilization process.
Most foodborne pathogenic bacteria are mesophilic they grow in a range of temperature similar to that of the human body.
NOM 121 SSA1 1994. Quesos Frescos
For this reason, it is important to prove the inhibition capacity of our films against aerobic mesophilic bacteria they are indicators of food contamination, but they are not pathogens in a strict sense. The control chitosan—starch film did not show any inhibitory effect on aerobic mesophilic bacteria.
These results mean that the addition of antioxidants significantly improved the antibacterial activity of the films, especially in the case of anthocyanins, since they are more stable than any other kind of antioxidants. The antibacterial activities of chitosan—starch films with natural antioxidants after 48 h is shown in Table 2 in accordance with section number 11—Test report of norm NOMSSA [ 19 ]—we had to report the bacterial growth at that time.
We observed a significant improvement in antibacterial activity due to the addition of natural extracts, and the same behavior was observed in the research of Duran et al. A similar result was obtained by Yuan et al. Paparella et al.
Section 2. The inhibition zone indicated for each film is related to the positive results shown during antimicrobial activity. Coliforms are Gram-negative bacteria that are indicators of possible food and water contamination; the main bacterium of this group is Escherichia coli.
The control chitosan—starch film showed little inhibitory effect on coliform bacteria only in one test. The other films showed medium antibacterial activity. The beetroot films showed the same results as QS2. These results reveal that the addition of anthocyanins and resveratrol significantly improved the inhibition effect on this group of bacteria.
The antibacterial activity of chitosan—starch films with natural antioxidants after 24 h is shown in Table 2 in accordance with section 11—Test report of norm NOMSSA [ 20 ]—we had to report the bacteria growth at that time.
In accordance with the research of Shen et al. See Section 2. Fungi are a huge group of microorganisms widely distributed in the environment. In this study, we observed inhibition activity against three kinds of fungi: Penicillum notatum , Aspergillus niger , and Aspergillus fumigatus. The Penicillum fungi and the Aspergillus fungi exist on most surfaces; they grow during food decomposition for example in bread and fruits.
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They are not potentially dangerous to humans except in those with weak immune systems. The control chitosan—starch film showed little inhibitory effect on coliform bacteria only in two tests. The resveratrol films showed medium antibacterial activity.