Edible anti-mold coating to control aflatoxin in pistachios

Abstract

This invention relates to a natural edible coating with anti-mold properties to reduce microbial load and fungal contamination, limit aflatoxin production during storage, and increase the shelf life of pistachios. The main components of the coating include silk fibroin (for film strength), chitosan (with antimicrobial properties), sugar beet extract (strong antioxidant), Tween 80 (emulsifier), and glycerol (softener). The process of making this coating includes extraction and purification of silk fibroin, dissolution of chitosan, extraction of active compounds from the sugar plant Tigal, and then optimal homogenization of the components and ultrasonic applications and creation of a nano-emulsion structure. The coating is applied to the pistachios by immersion method and dried at the appropriate temperature. This coating, by creating a physical barrier, prevents the exchange of moisture and oxygen and prevents the growth of mold. The inherent properties of chitosan and the active compounds of sugar extract of Tigal create direct antimicrobial properties against fungi and bacteria, which prevents the production of aflatoxin. Sugar extract of Tigal also prevents the oxidation of fats and the deterioration of the taste and smell of the product, which ultimately leads to an increase in the shelf life of pistachios.

Description

Description of the invention Title of the invention Edible anti-mold coating to inhibit aflatoxin in pistachios Technical background of the relevant invention The present invention is in the field of chemistry, food industry and agriculture, especially in the field of active packaging technologies and edible coatings, and methods for maintaining the quality and safety of agricultural products, nuts and dried fruits, especially pistachios. Technical problem and stating the objectives of the invention Mold and growth of toxin-producing fungi in the production and storage of nuts and dried fruits such as pistachios, almonds, walnuts, etc., is one of the most serious challenges. Mycotoxins are toxic secondary metabolites produced by some fungi, including Aspergillus, Penicillium, and Fusarium species, during the growth, harvesting, transportation, and storage stages of agricultural products. Among the most dangerous known mycotoxins are aflatoxins, especially aflatoxin B1, which, in addition to reducing the appearance, taste, and commercial value of the product, is considered a serious threat to consumer health due to its severe carcinogenic and mutagenic properties. Therefore, controlling fungal contamination and reducing aflatoxin production in agricultural products, especially in nuts and dried fruits, and especially pistachios, is of particular importance. According to existing reports, a significant percentage of agricultural products in the world are exposed to contamination by these toxins, which, in addition to threatening public health, also causes heavy losses to producers and exporters.Meanwhile, compliance with strict international standards, especially in export markets, has doubled the need to develop effective, safe, and economical methods for pollution prevention and control. Common methods for detoxifying mycotoxins are divided into three general groups: physical, chemical, and biological. Physical methods such as washing, screening, mechanical separation, irradiation, ultrasonic treatment, and the use of adsorbents are usually associated with limitations such as time-consuming, low efficiency, and limited effect. Chemical methods such as the use of hydrogen peroxide, acids and bases, ammonia, and other antifungal compounds, although effective in some cases, usually require special equipment, high cost, and difficult operating conditions, and may have an adverse effect on the sensory, appearance, and nutritional value of the product. Biological methods, despite their relative advantages, are often complex or costly for widespread industrial use. Therefore, the need for a natural, safe, effective and economical edible coating to protect pistachios against mold and reduce the risk of aflatoxin production is clearly felt. Accordingly, the aim of this invention is to produce a natural edible anti-mold coating based on silk fibroin, chitosan biopolymer and sugar beet extract that can act as a protective layer and prevent aflatoxin production while reducing mold growth. This edible coating reduces moisture and oxygen exchange by creating a physical barrier, thereby providing unfavorable conditions for the growth of fungi. Also, chitosan, due to its inherent antimicrobial properties, and sugar beet extract, due to its bioactive compounds, exert a direct inhibitory effect on the growth of fungi and some bacteria. In addition, the antioxidant property of sugar beet extract can prevent lipid oxidation, loss of taste and odor quality, and premature spoilage of the product. As a result, this invention, by utilizing natural and edible ingredients, will increase the shelf life of pistachios, maintain the sensory and nutritional quality of the product, reduce fungal contamination and aflatoxin production, and can be a suitable alternative to costly, chemical, and inefficient methods. A description of the state of the prior art and the history of developments related to the claimed invention. Due to the many applications and importance of edible coatings in industry, much research has been conducted in the field of making edible coatings from inexpensive raw materials, especially chitosan and other natural biopolymers. All natural polymeric materials can be suitable raw materials for preparing edible coatings. A search of patents registered in Iran in this field shows that there have been reports of the production of edible and antimicrobial coatings, but no application has been registered using a synergistic combination of three components with multiple functions, simultaneously combining physical protection, biological inhibition and oxidation prevention using edible silk cocoon proteins along with sugarcane extract and the natural biopolymer chitosan, which is suitable for several nut and dried fruit products. 1- In the application with registration number 117090, a system based on atmospheric pressure cold plasma has been designed and used in plasma technology to control, reduce and eliminate aflatoxin. Design and manufacture of a non-equilibrium plasma generator of the dielectric discharge type, in fact, active plasma species such as free radicals, ions, highly reactive species and ultraviolet radiation during the process by intense bombardment of microorganisms and spores with their cell walls through various mechanisms, causing inactivation or death of microorganisms. 2- In the application with registration number 38105, the process of increasing the shelf life of fresh pistachios using modified atmosphere packaging used oxygen, carbon dioxide, and nitrogen gases in polyethylene packaging to increase the shelf life of the outer shell of fresh pistachios. 3- In the application with registration number 389070133, the process of reducing aflatoxin in pistachios is with the help of microbial strains. In this study, the ability of reducing aflatoxin by two probiotic species Saccharomyces cerevisiae and Electrobacillus rhamnosus LBGG was evaluated in order to reduce the toxicity of aflatoxin in pistachios. The fixation of yeast and bacteria on aflatoxin-contaminated pistachios in order to reduce the toxin does not affect the quality characteristics such as color, texture and peroxide value. The results indicate that this feature can be effective in the direction of aflatoxin in pistachio storage terminals. 4- In the declaration with registration number 38710307, ​​the process of detoxifying pistachios from aflatoxin toxins and detoxifying plants from the above toxins used oxidizing agents and inert gases. 5- In the application with registration number 387111233, the production of an edible antimicrobial coating to prevent the growth of Aspergillus flavus soil in pistachios is made of a natural polymer based on edible whey concentrate and thyme extract. An edible antimicrobial coating is a type of packaging that can kill microorganisms or prevent their growth, and therefore increase the shelf life of perishable products and increase the health of packaged products. 6- In the application with registration number 3010371, the formulation of a solution increases the shelf life of fresh pistachios. By adding plant essential oil and zinc nanoparticles to packaged pistachios, microbial activity in the environment is simultaneously prevented. At the same time, the antioxidant activity of these two factors increases the life of the fresh skin and prevents its spoilage. In all registered applications and reviewed sources, in no case has a coating of three materials been formed that simultaneously provides a physical barrier, biological inhibition, and antioxidant protection in a single layer, and silk cocoon fibroin, biodegradable biopolymer materials such as chitosan, and sugarcane extract have been used. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention To solve the problem of mold and mycotoxin contamination of pistachios, especially aflatoxin, in this invention, a natural anti-mold edible coating has been designed and presented, which is prepared from a combination of silk fibroin, natural biopolymer chitosan, and sugar beet extract. This coating is used to create a protective layer on the surface of pistachios to prevent the growth of fungi and the production of mycotoxins while maintaining the appearance and sensory quality. In terms of structure and function, it offers an integrated and practical solution to deal with the problem of mold in pistachios, because it simultaneously covers three main needs: creating a physical barrier, microbial inhibition, and antioxidant protection. 1. Silk fibroin: As a film-forming substrate with the ability to form a transparent, thin, and strong layer on the surface of the pistachio, it creates a physical barrier against the penetration of oxygen and moisture. 2. Chitosan biopolymer: As a natural antimicrobial agent that inhibits the growth of fungi by disrupting their cell membranes. 3. Sugarcane extract: As a strong antioxidant and antifungal agent that, in addition to biologically inhibiting fungi, prevents the oxidation of fats and sensory spoilage of pistachios. The combination of these three materials forms a coating that simultaneously provides a physical barrier, biocontainment, and antioxidant protection in a single layer. In this invention, the nanoemulsion method was used to prepare the edible coating. First, the aqueous phase was prepared from a solution of chitosan and fibroin, and the optimal pH of the solution was adjusted so that the protonated groups of chitosan interacted with the hydroxyl groups of fibroin to the greatest extent. Then, the extract of sugar beet, Tween 80, and glycerol were added to the aqueous phase and stirred with a high-speed homogenizer for 10 to 15 minutes. The next step is the nanostructure formation using high-pressure ultrasonication, which is very important and vital, and must be pulsed to prevent the increase in ambient temperature, which causes denaturation of fibroin and destruction of the active compounds of sugar beet extract. The physicochemical, microbial, performance, and efficiency of the edible anti-mold coating were investigated over a period of 6 months.Thermal gravimetric analysis tests, which determine the drying time of coated pistachios in the oven, were performed on the coating prepared in solution and the dried coating, and the results showed that the prepared coating showed good thermal resistance in both cases (Figure 3a and b). Microbial mold counting tests were also conducted on uncoated (control) and coated pistachios at time intervals of 10, 45, 30, and 60 days, and the results obtained showed the effectiveness of the coating in preventing mold growth by up to 99% (Figure 4). Physical tests and structural examinations such as weight loss, water absorption, thickness, turbidity, and water vapor permeability of the coating were performed, and the results showed that the prepared coating was of high quality (Table 1). The most important test in examining the effectiveness of the coating in preventing and controlling aflatoxin was performed on healthy and contaminated pistachio samples.In this study, a pistachio sample was first obtained from the market and its aflatoxin content was examined using a high-performance liquid chromatography (HPLC) device, which was negative and the pistachios were healthy (Figure 5a). At the stage, the pistachios were divided into two groups: one group was uncoated and the other was coated pistachios. Then, the pistachios of both groups were contaminated with aflatoxin at a concentration of 20 ppb and the samples were placed in the refrigerator. Then, the aflatoxin content of the samples was examined at intervals of 2 and 7 days, and the results obtained showed that the aflatoxin content in the coated pistachios had decreased by 95% (Figure 5b) and the uncoated pistachio sample contained aflatoxin (Figure 5c).Elasticity, puncture resistance, and stretchability were also measured using a texture analyzer on the anti-mold coating, and the results showed very high quality of the coating in terms of elasticity percentage and resistance to puncture and impact (Figure 2). To better examine the structure of the coating in absorbing aflatoxin, electron microscope images were prepared for coated and uncoated samples, which showed good nanostructure images in trapping aflatoxin (Figure 6a and b). To further examine the effectiveness of the coating in preventing mold, the optimized formula was tested on fruits (persimmon, tomato, apple, cucumber, etc.), and the results showed that the coated fruits maintained their quality in terms of appearance, mold, and taste over a period of 20 days. Explanation of shapes, maps and diagrams Figure 1: Infographic of how to prepare an anti-mold edible coating A clear and precise statement of the advantages of the claimed invention over prior inventions. So far, there has been no report on the production of an edible anti-mold coating from sugar beet extract with silk fibroin and chitosan biopolymer, and the above-mentioned inventions have been mostly performed on fresh green-skinned pistachios. There has also been no research on the use of sugar beet extract as a strong antioxidant and antifungal agent that, in addition to biologically inhibiting fungi, prevents lipid oxidation and prevents sensory spoilage. What is presented in this application is the formation of a coating of three materials that simultaneously provides a physical barrier, biological inhibition and antioxidant protection in one layer, which has the following advantages: 1. Synergistic effect in antimicrobial activity: Most conventional anti-mold coatings are either based on chemicals (such as sodium benzoate or potassium sorbate) or use only one polymer. Chitosan alone destroys the cell wall of fungi. Sugarcane extract contains polysaccharide and phenolic compounds that have mold-inhibiting properties. The combination of these two with the protein structure of silk fibroin creates a controlled release system that allows the anti-mold property of the coating to be maintained for a longer period of time and not to be lost quickly. 2. High barrier properties: Conventional coatings often have poor resistance to moisture exchange with oxygen, which in turn causes mold, but silk fibroin has a highly crystalline and orderly structure. When this protein is combined with chitosan, it creates a dense polymer network that reduces the permeability to water vapor and oxygen. This means that the internal environment of the pistachio under the coating remains so dry and oxygen-free that mold growth becomes virtually impossible. 3. Increased anti-mold activity: When sugarcane extract is transformed into nanoparticles, its contact surface with pathogens (molds) increases significantly and its antimicrobial effect multiplies. 4. Biocompatibility and Edibility: Many existing industrial coatings contain synthetic preservatives that may be allergenic to the consumer or change the taste of the pistachio. However, all components of this coating (silk fibroin, chitosan, and sugar beet) are derived from natural sources and are completely biodegradable and edible. 5. Improving appearance: Old coatings often cause the product to become cloudy or the surface of the pistachio to stick. However, the nano-emulsion method makes the layer formed on the pistachio very thin, transparent, and shiny, and spreads evenly in all areas, leaving no empty holes on the surface of the pistachio. This feature gives the pistachio a fresh and customer-friendly appearance due to its natural shine. 6. It is economically viable for industrial units and can help reduce waste, increase product shelf life, and improve export quality. Description of at least one implementation method for implementing the invention The method of synthesizing the edible coating includes: 1. Preparation of chitosan solution: Dissolve 1 to 2.5 grams of chitosan powder in 100 ml of dilute acetic acid. The mixture is placed under a magnetic stirrer for 24 hours at room temperature until a clear and uniform solution is obtained. The pH of the solution is adjusted to about 5 to 5.5 using sodium hydroxide. 2. Extraction and preparation of silk fibroin: We boil natural silk fibers in a 0.02 M sodium carbonate solution for 30 minutes to remove sericin, then we wash the resulting fibers with distilled water and dry them. We dissolve pure fibroin in a suitable solvent at a temperature of 60 ° C, then we dialyze the solution for 48 hours. We dissolve the pure fibroin solution in distilled water with a concentration of 0.5 to 2%. 3. Preparation of sugarcane extract: Dissolve 10 grams of dried and ground sugarcane powder in 100 ml of hydroalcohol. The mixture is stirred for 24 hours, then the extract is filtered and the concentrated extract is obtained by evaporating the solvent. 4. Preparation of the final formulation: To one volume of the stirred chitosan solution, add about half the volume of the silk fibroin solution, then add twice the volume of concentrated sugar extract of sugarcane to the mixture, then add between 0.2 and 1% glycerol as a plasticizer and about 0.1 to 0.8 Tween 80 to it, and homogenize the mixture with a high-speed homogenizer for 5 to 15 minutes to create aggregates of discrete particles and uniform dispersion. The homogenized suspension is subjected to an ultrasonic process for 10 to 30 minutes. During the ultrasonic process, the sample temperature is maintained between 20 and 30 degrees with ice to prevent the destruction of bioactive compounds. The ratio of sugar extract to glycerol should be adjusted to prevent coating adhesion due to the trehalose sugars of sugar. The pH of the chitosan solution and fibroin solution when combined is very important and should be adjusted so that the protonated groups of chitosan interact more with the hydroxyl groups of fibroin to prevent rapid dissolution of the film and formation of precipitates. 5. Applying coating to pistachios: Cleaned and dried pistachios are immersed in the coating solution for 40 to 60 seconds, then the pistachios are removed and the excess solution is removed. Then the samples are dried at a temperature of 35 to 50 degrees until the coating layer is stabilized. In the final optimized formula, the ratio of chitosan: fibroin: sugar beet extract is 1: 0.5: 2, respectively. Explicit mention of the industrial application of the invention This invention can be used in processing and packaging units for nuts and dried fruits such as pistachios, almonds, walnuts, raisins, hazelnuts, dates, etc., agricultural export industries, warehouses and cold storage for food. This process can be carried out on laboratory, semi-industrial and industrial scales and using common equipment such as mixing tanks, spray devices, immersion devices, hot air dryers and packaging lines.

Claims

Claims What is claimed: Claim 1) Edible anti-mold coating consisting of bio-based materials to reduce microbial load and inhibit the growth of aflatoxin-producing fungi and increase the shelf life of pistachios, including: silk fibroin, chitosan biopolymer, and sugar beet extract. Claim 2) Edible coating according to claim 1, in which the weight ratios of the components: *Silk fibroin: between 0.5 and 2% by weight *Chitosan: between 1 and 2.5% by weight *Tighal sugar extract: between 2 and 4% by weight *Tween 80 emulsifier: between 0.2 and 0.8% by weight *Glycerol: between 0.2 and 1% by weight *Aqueous solvent to the remaining amount up to 100% by weight Claim 3) According to claim 1: Silk fibroin acts to create a basic film-forming structure and physical barrier, and the effective range for the balance of mechanical strength and permeability is less than 0.5 loose and more than 2% cracked. Claim 4) In accordance with claim 1, chitosan, with its antimicrobial properties, inhibits the growth of aflatoxin-producing fungi. The optimal range for maintaining antimicrobial properties without changing taste and texture is between 1 and 2.5%. Claim 5) In accordance with claim 1: Sugarcane extract prevents oxidative stress and further growth of microorganisms due to its antioxidant and antifungal compounds, and the effective range for inhibiting fungi under long-term storage conditions is between 2 and 4%. Claim 6) In accordance with claim 1: Glycerol is used as a plasticizer to increase flexibility and prevent cracking, and Tween 80 is used as an emulsifier and stabilizer to evenly distribute the sugar beet extract in the aqueous phase. Claim 7) The production method according to claim 1 includes dissolving fibroin in distilled water (optimal concentration), dissolving chitosan in dilute acetic acid (optimal concentration), then mixing both solutions and adjusting the optimal pH, adding sugar extract, tween and glycerol, and homogenizing the components with a high-speed homogenizer until a uniform solution is formed, and finally placing under ultrasonic waves to reduce particle size and create a nano-emulsion. Claim 8) In accordance with claim 1: Applying an anti-mold coating by immersion on pistachios for 40 to 60 seconds until a uniform layer is created on the surface and finally drying the pistachios at a temperature of 35 to 50 degrees Celsius. Claim 9) The coating prepared according to claim 1: with anti-mold properties is used to reduce microbial load, reduce fungal contamination and limit aflatoxin production during storage to increase shelf life, maintain sensory characteristics, color, flavor and texture of pistachios.

Images