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Development of Bioactive Opuntia ficus-indica Edible Films Containing Probiotics as a Coating for Fresh-Cut Fruit

Bioactive edible films have received more attention in recent years as a method for food preservation with value-added functions. The aim of this study was to develop a bioactive edible film containing mucilage of cactus (Opuntia ficus-indica) and incorporating the probiotic strain Enterococcus faecium FM11-2 as an active component to promote consumer health benefits. Opuntia ficus-indica is rich in nutritional and bioactive compounds and the abundance of this cactus makes it attractive for food applications. Mucilage of Opuntia ficus-indica contained 0.47 ± 0.06 mg/g total sugar, 0.33 ± 0.06 mg AGE/mL phenolic content, 0.14 mg/ mL vitamin C, and possessed 35.51 ± 1.88% DPPH scavenging activity. The edible film that was developed exhibited the following characteristics: thickness of 0.02–0.11 mm, percent moisture content 0.19–0.24%, water solubility 30.66–59.41% and water vapor permeability of 0.15–1.5 g·mm/m2·min·kpa, while the range of the variation depended on the type of plasticizer used (either sorbitol or glycerol). The addition of sorbitol in the film provided the maximum mechanical strength based on the evaluation of tensile strength, Young’s modulus and elongation at break (44.71 ± 0.78 MPa, 113.22 ± 0.23 MPa and 39.47 ± 0.61%, respectively). The optimal formulation of the edible film, according to the physicochemical, physical and maintenance of fresh-cut apple slices, contained cactus mucilage, gelatin, glycerol and a probiotic. The incorporation of a probiotic into the cactus film created a bioactive edible film that could provide a health benefit. While improvement is needed to maintain the survival rate of the probiotic, this work presents an exciting method for furthering the study of food preservation with edible films.

Introduction
Active food packaging films have been developed to be eco-friendly, enhance product shelf life and meet the demand for healthier foods. Recently, there have been dramatic improvements in the development of edible films and biodegradable films due to the growing interest in reducing environmental pollution caused by global plastic usage. It has been suggested that food plastics and wrappers account for over half the world-wide plastic usage. Edible films act as a barrier between the covered product and the surrounding medium to delay the migration of moisture, gases and lipids. More recently, active edible films have been developed to carry more additives such as nutrients, flavors, antimicrobials, oxygen scavengers, antioxidants, antimicrobials, ethylene scavengers, and moisture absorbers [1]. The principle components for producing edible/biodegradable films are film forming biopolymers, such as carbohydrates, proteins, a solubilizing medium (water, Polymers 2022, 14, 5018 3 of 16 developed bioactive edible film as a coating on fresh-cut apple slices and determined the freshness, weight loss and probiotic survival rates. This study provides insight for the further exploration of ingredients and physicochemical/mechanical properties for the development of bioactive edible coatings. These finding can now be applied to develop more variations in films for use with fresh-cut fruits and vegetables for health benefits and food preservation in the future.

Cactus Mucilage Extraction
Opuntia ficus indica were purchased from Chang Phueak district, Chiang Mai province, Thailand. The size of the leaves ranged between 30 and 40 cm in length, with an age of approximately 2 years. Fresh pads were washed with distilled water and the thorns were removed. The outer layer was removed, while leaving the interior white flesh intact. The extraction of mucilage from cactus pads of OFI was performed according to the method of Rim et al. (2018) and Otálora et al. (2021) with modification [18,19]. The interior white flesh was cut into small pieces and placed in a 1000 mL beaker with distilled water 1:2 v/v (flesh: distilled water). The mixture was left at room temperature for 24 h and then the extract was filtered through nylon cloth. The extract was centrifuged at 12,880× g force at 4 • C for 30 min to remove the remaining particles. Subsequently, 95% v/v ethanol was added to the extract at a ratio of 1:2 v/v (extract: 95% v/v ethanol) for one night at 4 • C to precipitate the mucilage. The ethanol was then completely evaporated in a water bath set to 50 • C. The residue was collected in a 1000 mL Duran flask for further application.

Analysis of the Composition of Cactus Mucilage 2.2.1. Total Sugar Content
Total sugar content of the cactus mucilage was evaluated using the phenol-sulfuric method, which is a simple and rapid colorimetric method that uses Absorbance at OD 490 using a spectrophotometer [20]. The concentration range of the glucose standard solution was 0-0.2 mg/mL. A 10-fold dilution of the cactus mucilage was evaluated and compared to the glucose standard curve to obtain the total sugar content.

Total Phenolic Content
The total phenolic content of cactus mucilage was determined using the Folin Ciocalteu method from Rim et al., 2018, with modification [18]. The standard curve was prepared using a gallic acid (Sigma-Aldrich, St. Louis, MO, USA) solution at concentrations of 0-0.3 mg/mL. The reactions were performed by pipetting 500 µL of each concentration of gallic acid solution into test tubes, followed by the additions of 5 mL of Folin Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) (10%) and 4 mL of 7% w/v sodium carbonate solution. The reactions were mixed after the addition of each reagent and then incubated for 15 min in the dark. The absorbance was monitored at OD 765 . To measure test samples, aliquots of 500 µL of mucilage were mixed with 5 mL Folin Ciocalteu reagent and 4 mL of 7% w/v sodium carbonate following the standard protocol. The absorbance was measured at 765 nm and the results were expressed as mg of gallic acid equivalent per g of mucilage (mg GAE/g MUC).

Vitamin C Content
Vitamin C content was determined according to the method of Nantawan et al. (2017) with some slight modifications [21]. Cactus mucilage (2 mL) was added to an Erlenmeyer flask containing 100 mL of distilled water. Next, 5 mL of 1% w/v starch solution was added. The mixture was shaken and titrated with an iodine solution to the end point. The volume of iodine solution used was recorded and was used to calculate the vitamin C content using the triangular law calibration. The concentration of 1 mg/mL of ascorbic acid (Sigma Aldrich, St. Louis, MO, USA) solution is equivalent to the use of 13.6 mL of iodine solution for the titration. If 1 mL of iodine solution was used, the content of ascorbic acid was 0.074 mg/mL. Therefore, the ascorbic acid content of the cactus mucilage was calculated from: where: A is the amount of ascorbic acid in the sample solution (mg) V I2 is Volume of iodine used for titration (mL)

Radical Scavenging Effect
Free radical scavenging was determined using the DPPH radical scavenging method. First, 0.1 mM of methanol-2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, St. Louis, MO, USA) was prepared in methanol and the absorbance was measured with a spectrophotometer at 517 nm for the determination of the percentage inhibition. Then, 0.5 mg/mL cactus mucilage was prepared in distilled water and 1 mL was pipetted into test tubes, followed by the addition of 2 mL of 0.1 mM of DPPH solution. The reaction was mixed well and set aside in the dark for approximately 30 min, after which the absorbance was measured at OD 517 . Experiments were repeated three times, and the precent inhibition was calculated using the following equation: where A sample is the absorbance of the sample + DPPH A DPPH is the absorbance of DPPH

Preparation of Probiotic Cells
E. faecium FM11-2 was isolated from Thai fermented chicken in our previous work [22]. The strain was streaked on MRS (deMan, Rogosa, Sharpe, Oxoid, Basingstoke, UK) agar and incubated at 37 • C for 48 h prior to culture in MRS broth for another 48 h until the OD 600 reached approximately 1.0. The suspended culture was then ascetically transferred into eppendorf tubes (1 mL per tube) and centrifuged at 2060× g force at 4 • C for 5 min to collect the cell pellets. The supernatant was discarded, and the cell pellets were washed twice (1 mL per wash) with 0.85% sodium chloride (NaCl) solution and resuspended at a concentration of 1.2 × 10 8 CFU/mL prior to incorporation into the film.  Table 1 to ensure homogenous mixing. The prepared mixtures were cast onto plastic Petri dishes (total volume of 15 mL) and dried in a ventilated oven at 45 • C for 24 h to form a film. The resulting films were stored in a desiccator at 25 • C and 53% RH for 48 h prior to testing. RH was monitored using a psychrometer based on a dry and a wet temperature sensor. To incorporate the probiotic (E. faecium FM11-2), the mixtures were aseptically prepared as described above, after which the solution was cooled to 37 • C and the probiotic was added at a concentration of 1.2 × 10 8 CFU/mL. The films were dried at 35 • C for 24 h under aseptic conditions. Additionally, 50 mL of the solution was kept for dipping fresh cut apple for further analysis. The thickness of the film was evaluated using a micrometer (Mitutoyo, Kawasaki, Kanagawa, Japan) with measurements taken at 5 arbitrary locations and the mean value of the films was calculated [23].

Moisture Content
The moisture content of the films was determined using a modification of the protocol by Rim et al. (2018) [18]. The film samples were cut into pieces with an initial weight (m i ) of 0.5 g and then oven dried at 105 • C until the weight was stable to obtain the final weight (m f ). The moisture content of the film was calculated based on the formula: where %MC is the percent moisture content of the film, m i is the initial weight of the film (g) and m f is the weight of the film after curing to a constant weight (g).

Film Solubility
Film solubility was analyzed according to the method of Jouki et al. ( 2013) with some modifications [24]. Film samples of 0.5 g were oven dried at 105 • C until constant weight was achieved (m i ) and then cooled to room temperature. The samples were then submerged in 50 mL of distilled water at room temperature for 30 min. The undissolved matter was collected from the liquid and oven dried at 105 • C until the weight was constant (m f ). The water solubility of the film is reported as a percentage of water solubility (% water solubility) and calculated using the equation:

Water Vapor Permeability
Water vapor permeability of the film was analyzed according to the ASTM E96/E96M (2015) standard method with some modifications. The experiment was conducted using the desiccant method. The film samples were sealed on an open cup containing 15 g of pre-dried silica gel and incubated at room temperature (35 • C and 60% RH) until the weight was stable [25]. Water vapor transmission is measured by weight gain (Weight change as function of time) and calculated using a linear regression equation (R 2 > 0.99) and water vapor permeability (g·mm/m 2 ·min·kPa): where: WVTR (g/m 2 ) is the water vapor permeability rate. It is obtained as the slope (g/min) divided by the transmission area (m 2 ). X (mm) is the film thickness. ∆p (kpa) is the difference in water vapor pressure between the films (∆p = p(RH 2 − RH 1 ) = 337.60 where p is the saturation pressure of water at 35 • C, RH 2 = 60% and RH 1 = 0%)

Mechanical Properties
Mechanical testing of the film was performed by Texture Analyzer model TA-XT plus C (Stable Micro Systems, Godalming, UK) to assess tensile strength, the elongation at break, and the Young's modulus (stress-strain) in accordance with the ASTM D882 (2001) standard test method. Each film sample was cut to a size of 15 mm × 60 mm and a test speed of 100 mm/min was used. The values of tensile strength, elongation at break and Young's modulus are displayed using the program Exponent.

Properties of Film Coating on Fresh-Cut Apple
The apple was cut into pieces and soaked in 1.5% w/v saline solution for 10 min to prevent the formation of melanin that can cause a browning reaction on apple pulp. The samples were then dipped in film formulations 5 and 6 containing probiotics ( Table 1). The dipped apples were left to dry on a sieve for 10 min. The samples were then refrigerated at 4 • C and the weight loss, analysis of the change in appearance and the viability of the probiotic were analyzed on days 0, 2, 5 and 7. To determine the survival of probiotics on the coated apples, 10 g of the coated apple sample was placed in a bag containing 90 mL of 0.85% saline. The sample was dissociated with a Stomacher, and then diluted to the desired level. The spread plate technique was used to inoculate MRS Agar medium and plates were incubated at 37 • C for 2 days. The colonies formed on the medium were reported as log CFU/g.

Statistical Analysis
All experiments were performed in triplicate and the data was statistically analyzed by ANOVA with Duncan's multiple range to compare between the different treatments.

Results and Discussion
The complex nature of cactus mucilage makes it a valuable resource for nutritional and health benefits [26]. Previous studies have found that ethanol facilitates the extraction of phenolic compounds and antioxidant compounds from OFI, so we used this methodology to direct our studies. Our results determined levels of total sugar, phenolic content, vitamin C and DPPH radical scavenging for the extracted cactus mucilage, as shown in Table 2. Preservation of active groups, such as phenolic acids, is key in creating a useful biological packaging film and their antioxidant potential is involved in many health benefits such as prevention of inflammation, cardiovascular dysregulation, and neurodegenerative diseases [27]. Additionally, antioxidant activity can depend on the presence of vitamin C, therefore we evaluated the DPPH radical scavenging activity of the extracted cactus mucilage. Our results indicate that our extraction method preserved nutritional value and active compounds that may promote health in combination with the bioactive edible film. The appearance of the edible films among the 6 formulas ( Figure 1) differed based on the plasticizer used. Both glycerol and sorbitol are suggested as good plasticizers for gelatin, as they can reduce intermolecular hydrogen bonding in gelatin and increase intermolecular spacing. It is postulated that the high molecular weight of sorbitol creates larger intermolecular spacing and leads to better film flexibility [28]. In our study, the films with glycerol as a plasticizer were rough and brittle, whereas the films with sorbitol were smooth and tough. The brittleness of film plasticized with glycerol can be explained due to its low molecular weight of 92 g/mol, in comparison with 182 g/mol for sorbitol [29,30].
The appearance of the edible films among the 6 formulas ( Figure 1) differed based on the plasticizer used. Both glycerol and sorbitol are suggested as good plasticizers for gelatin, as they can reduce intermolecular hydrogen bonding in gelatin and increase intermolecular spacing. It is postulated that the high molecular weight of sorbitol creates larger intermolecular spacing and leads to better film flexibility [28]. In our study, the films with glycerol as a plasticizer were rough and brittle, whereas the films with sorbitol were smooth and tough. The brittleness of film plasticized with glycerol can be explained due to its low molecular weight of 92 g/mol, in comparison with 182 g/mol for sorbitol [29,30].  Physicochemical properties determine the ability of the edible films to maintain the integrity of packed products. Critical parameters are the strength of the material, including the film thickness, percent moisture content, percent film solubility and WVP [31]. Film formula 6 contained cactus mucilage, gelatin, sorbitol and probiotic and was the thickest of our formulations (thickness 0.11 ± 0.04 mm) ( Table 3). The addition of the probiotic and sorbitol had a significant effect on the thickness of the edible film, and it has been shown that film thickness is mostly related to the concentration of solid matter and the viscosity of solutions. Additionally, there are reports that the addition of a probiotic and the number of solutes incorporated could increase the solid content of the film forming solution [32,33]. Furthermore, with a constant amount of gelatin and plasticizer, the film thickness of highviscosity solutions was thicker than that of low-viscosity solutions because of the influence of water vapor permeability [34,35]. In the previous work, films formulated with OFI mucilage and additives including glycerol, polyethylene and glycol-oleic acid, exhibited increased film thickness (to 0.222 mm) [13]. The moisture content and film solubility are important factors in determining the melting of edible films in the mouth [16]. In the present study, the presence of probiotics and the addition of either of the two different plasticizers caused no significant effect on film moisture. Similar results were found in previous studies with the application of probiotics [36]. Moisture content of the films composed of glycerol were in the range of 0.21-0.36%. In the presence of a constant amount of polymer, the films composed of glycerol generally retained more moisture than the films composed of sorbitol (range of 0.19-0.21%). This finding correlated well with the results in the literature [37]. While the combination of probiotic and sorbitol seemed to be the optimal formula for the edible film based on film thickness and percent moisture content, the percent film solubility was only 30.66%. Therefore, the probiotic with cactus mucilage using glycerol as a plasticizer would be a more suitable edible film due to higher solubility and less cohesion in the polymer matrix. This should lead to a preferable mouth feel. Although edible films and coatings may not completely replace external food packaging, they can provide a good barrier to oxygen and water, which can prolong food stability by reducing the rates of moisture and gas transfer between food and the surrounding environment [31]. Previous studies have shown that moisture plays a key role in food spoilage, and, therefore, to be a useful component for food storage, edible films must create a low enough water vapor barrier to circumvent the issue [38,39]. The film must not only exclude atmospheric water from the product, but also prevent water loss from the food that is wrapped [40,41]. In our study, bioactive edible films made with sorbitol exhibited a higher WVP value (0.16-1.50 g·mm/m 2 ·min·kpa) when compared with those made with glycerol (0.05-0.31 g·mm/m 2 ·min·kpa). This higher permeability and moisture loss may make sorbitol a less desirable plasticizer for food preservation (Table 3). Additionally, of the two different plasticizers tested (glycerol and sorbitol), sorbitol films were less fragile and probably not applicable for bioactive edible films (due to mouth feel) (Figure 1). Percent film solubility when glycerol was used as a plasticizer was approximately 53-61%, while that of sorbitol was 31-45%. The higher solubility is important because the coating would be consumed with the product as an edible matrix [41]. Our results indicate glycerol-based plasticizers are more applicable for use in formulations of OFI edible films. Table 3. Film thickness, percent moisture content, percent solubility and water vapor permeability of the 6 film formulas. The different letters represent a significant difference of p < 0.05. The error bar indicates the standard deviation (n = 3). Tensile properties of the films, including tensile strength, Young's modulus and elongation at break, were measured and presented in Figures 2-4, respectively. The use of either glycerol or sorbitol as a plasticizer for neat gelatin had no significant difference on tensile strength. However, incorporation of sorbitol enhanced the tensile strength when gelatin was mixed with cactus mucilage. The film (formula 4) prepared from mixtures of gelatin, cactus mucilage, and sorbitol had the highest tensile strength (50 MPa), while the addition of a probiotic had no significant effect (45 MPa). The same trend was noticed in the films plasticized with glycerol. From this we conclude that the addition of probiotic had no significant effect on the tensile strength. Young's modulus of the films containing glycerol was below 40 MPa, while that of the films containing sorbitol increased significantly to a value higher than 100 MPa. Young's modulus of the film prepared from the mixture of gelatin, cactus mucilage and sorbitol (formula 4) was 183 MPa, and it dropped significantly to 113 MPa when probiotic was added (formula 6). The increase in tensile strength and Young's modulus might be due to the presence of more hydroxyl groups in sorbitol compared to glycerol. Interaction between sorbitol and other components such as gelatin and cactus mucilage extracts could occur through hydrogen bonding. The highest elongation at break of 45% was found for the film prepared from the mixture of gelatin, cactus mucilage and glycerol (formula 3) and the elongation decreased to 23% when probiotic was incorporated (formula 5). The film prepared from gelatin, cactus mucilage and sorbitol (formula 4) exhibited 23% elongation at break, which increased to 39% when probiotic was added (formula 6). Overall, these results suggest that the addition of sorbitol tended to provide better mechanical properties, in comparison to glycerol. Film formula 6 exhibited the most robust mechanical properties based on tensile strength, Young's modulus and elongation at break (44.71 ± 0.78 MPa, 113.22 ± 0.23 MPa and 39.47 ± 0.61%, respectively). These characteristics indicate that this film might be better suited for food wrapping applications, rather than as an edible film component.

Formula
We also evaluated the influence of the edible coatings on fresh-cut apple slices based on physicochemical parameters including the freshness and weight loss after refrigeration at 4 • C on days 0, 2, 5 and 7. In vegetables, weight reduction during storage occurs by the loss of dry matter through respiration and by the loss of water due to transpiration. Thus, the physiological activity of fruits/vegetables during storage promotes the degradation of compounds, reduction in appearance, and quality loss which can cause rejection of the product by consumers [42]. Our results ( Figure 5) indicate that control samples without an edible coating exhibited characteristics which make them unlikely for consumption. In contrast, the samples coated with any of the tested formulas still partially maintained their freshness and extended the shelf life of the fresh-cut apples.      We also evaluated the influence of the edible coatings on fresh-cut app on physicochemical parameters including the freshness and weight loss afte at 4 °C on days 0, 2, 5 and 7. In vegetables, weight reduction during storag loss of dry matter through respiration and by the loss of water due to trans loss of dry matter through respiration and by the loss of water due to transpiration. Thus, the physiological activity of fruits/vegetables during storage promotes the degradation of compounds, reduction in appearance, and quality loss which can cause rejection of the product by consumers [42]. Our results ( Figure 5) indicate that control samples without an edible coating exhibited characteristics which make them unlikely for consumption. In contrast, the samples coated with any of the tested formulas still partially maintained their freshness and extended the shelf life of the fresh-cut apples. (Sorbitol + Cactus mucilage)

Formula 6
(Sorbitol + Cactus mucilage + Probiotic) Figure 5. The appearance of fresh cut apple slices dipped in edible coating compared to the control (uncoated) after refrigeration at 4 °C on days 0, 2, 5 and 7.

Formula 6
(Sorbitol + Cactus mucilage + Probiotic) Figure 5. The appearance of fresh cut apple slices dipped in edible coating compared to the control (uncoated) after refrigeration at 4 °C on days 0, 2, 5 and 7.
In this study, the weight loss of fresh-cut apples with and without edible coatings was also evaluated ( Figure 6). Significant weight loss occurred in uncoated fresh-cut apples (control). Constant losses in weight were observed in all coated samples up to day 7, except for samples that were coated with the bioactive edible formula 5, which exhibited no significant reduction in weight at the end of 7 days of storage. Bioactive edible formula 6 also exhibited a protection against weight loss, though not to the same degree as formula 5. Previous studies reported weight loss of stored product was lower when yeasts of the species Cryptococcus laurentii were added in the coatings, suggesting that the addition of microorganisms can influence this aspect of food preservation [43]. Our result also suggested the lower WVP of formula 5, compared to formula 6 ( Table 3), may help to counter water loss from the product. Reduction in product weight loss and darkening, as well as maintaining the freshness and extending the shelf life of fresh-cut products, are all In this study, the weight loss of fresh-cut apples with and without edible coatings was also evaluated ( Figure 6). Significant weight loss occurred in uncoated fresh-cut apples (control). Constant losses in weight were observed in all coated samples up to day 7, except for samples that were coated with the bioactive edible formula 5, which exhibited no significant reduction in weight at the end of 7 days of storage. Bioactive edible formula 6 also exhibited a protection against weight loss, though not to the same degree as formula 5. Previous studies reported weight loss of stored product was lower when yeasts of the species Cryptococcus laurentii were added in the coatings, suggesting that the addition of microorganisms can influence this aspect of food preservation [43]. Our result also suggested the lower WVP of formula 5, compared to formula 6 ( Table 3), may help to counter water loss from the product. Reduction in product weight loss and darkening, as well as maintaining the freshness and extending the shelf life of fresh-cut products, are all important factors for the commercialization of edible films. Our results indicated bioactive edible formula 5 was the best suited to meet these parameters. important factors for the commercialization of edible films. Our results indicated bioactive edible formula 5 was the best suited to meet these parameters. The global market for probiotics has greatly expanded in recent years guided by the rising demand for healthy diets and wellness. Probiotics can be delivered either through conventional means via pharmaceutical related products or non-conventional methods such as in food-based products. Probiotics have been added to several food products as well as incorporated into biopolymeric matrices to develop active food packaging. This packaging serves as an alternative method for controlling foodborne microorganisms, improving food safety, and providing health benefits [31]. In our study, probiotic E. faecium isolated from fermented chicken was incorporated into cactus mucilage containing either glycerol or sorbitol as a plasticizer. We evaluated the probiotic strain in films to assess The global market for probiotics has greatly expanded in recent years guided by the rising demand for healthy diets and wellness. Probiotics can be delivered either through conventional means via pharmaceutical related products or non-conventional methods such as in food-based products. Probiotics have been added to several food products as well as incorporated into biopolymeric matrices to develop active food packaging. This packaging serves as an alternative method for controlling foodborne microorganisms, improving food safety, and providing health benefits [31]. In our study, probiotic E. faecium isolated from fermented chicken was incorporated into cactus mucilage containing either glycerol or sorbitol as a plasticizer. We evaluated the probiotic strain in films to assess stability in terms of bacterial cell viability. First, we studied the viability of the probiotic after submerging fresh-cut apple slices in solutions containing cactus mucilage and plasticizer (either glycerol or sorbitol) and the probiotic bacteria (10 6 -10 8 CFU·g −1 ). The viability of the probiotics on the coated fresh cut apple slices dropped from the initial population of 10 6 -10 8 CFU·g −1 (on Day 0) to 10 3 CFU·g −1 on day 7 after refrigeration at 4 • C (Figure 7). The film formula, processing temperature for the film distribution and the storage conditions have all been found to influence the viability of the probiotic. Although there is no consensus among the international scientific community about effective probiotic doses to achieve health effects, researchers have suggested minimum doses between 10 6 and 10 9 CFU·g −1 to ensure therapeutic effects [44]. Other studies have shown probiotic viability in pure pullulan film was around 80% after 10 days of storage at 25 • C, decreasing to 35% after 20 days. Films incorporated with starches, however, show a correlation of decreased cell viability as the starch content increases [16]. Rice starch and proteins were observed to act synergistically to promote Lactobacillus rhamnosus GG cell viability [45]. Moreover, some factors such as WVP, oxygen content, water activity (a w ), heat-induced injuries, types of probiotic strains and mechanical stress can have adverse effects on the viability of probiotics in films [32]. For example, the addition of fructo-oligosaccharides (FOS) as a prebiotic and polysorbate 80 as a plasticizer together with glycerol and sodium alginate in linseed mucilage have been reported to maintain the number of the probiotic Lactobacillus casei LC-01 for 15 days [17]. Inulin, chitosan oligosaccharide (COS), galacto-oligosaccharide (GtOS) and FOS have been added to maize starch-based edible films for prebiotic purposes and have been shown to promote the growth of the probiotic bacteria Bifidobacterium infantis ATCC 15697 and Lactobacillus fermentum ATCC 9398 [46]. We hypothesize that the improvement in cell viability in those studies might be attributed to compounds in the films that promoted cell metabolism. Additionally, the conditions of film creation/drying and the storage condition might also be crucial for improvements in probiotic viability. Further study and process improvement for the incorporation of probiotics into bioactive edible films is required in our study. in linseed mucilage have been reported to maintain the number of the probiotic Lactobacillus casei LC-01 for 15 days [17]. Inulin, chitosan oligosaccharide (COS), galacto-oligosaccharide (GtOS) and FOS have been added to maize starch-based edible films for prebiotic purposes and have been shown to promote the growth of the probiotic bacteria Bifidobacterium infantis ATCC 15697 and Lactobacillus fermentum ATCC 9398 [46]. We hypothesize that the improvement in cell viability in those studies might be attributed to compounds in the films that promoted cell metabolism. Additionally, the conditions of film creation/drying and the storage condition might also be crucial for improvements in probiotic viability. Further study and process improvement for the incorporation of probiotics into bioactive edible films is required in our study.

Conclusions
We developed an edible coating for fresh-cut apple slices that resulted in improved preservation and shelf life of the product. We identified cactus mucilage, gelatin, glycerol and E. faecium as a probiotic (formula 5) as the optimal formula for an edible film, accord-

Conclusions
We developed an edible coating for fresh-cut apple slices that resulted in improved preservation and shelf life of the product. We identified cactus mucilage, gelatin, glycerol and E. faecium as a probiotic (formula 5) as the optimal formula for an edible film, according to parameters which analyzed physicochemical and physical properties. A number of studies in the last decade have focused on the incorporation of probiotics and other active ingredients to create bioactive edible films, leading to the emergence of new and exciting technology. From our study, the probiotic film and coatings played a significant active role in extending food stability by minimizing the weight loss and maintaining the freshness of fresh-cut apple slices. Improvements are still required for maintaining the number of surviving probiotic microbes in our edible film during food storage. Future plans for the improvement of our film will include the addition of prebiotics, the modification of the plasticizers and utilization of other probiotic strains. We hope that edible films composed of cactus mucilage and a probiotic may be a novel functional food that could replace supplement intake. Our current findings are an exciting first step toward modification of bioactive edible films/coatings for use with fresh-cut fruits.


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Association between age at first calving, first lactation traits and lifetime productivity in Murrah buffaloes

Objective This study was conducted to estimate the association of age at first calving (AFC) with first lactation traits as well as lifetime performance traits in Murrah buffaloes. Methods Data on first lactation and life time performance of Murrah buffaloes (n = 679), maintained at Indian Council of Agricultural Research-Central Institute for Research on Buffaloes, Hisar, India during the period 1983 through 2017, were deduced to calculate heritability estimates, genetic and phenotypic correlation of different first lactation and lifetime traits. The univariate animal model was fitted to estimate variance components and heritability separately for each trait, while bivariate animal models were set to estimate genetic and phenotypic correlations between traits under study. Results The heritability was high for first peak milk yield (FPY, 0.64±0.08), moderate for AFC (0.48±0.07) and breeding efficiency (BE 0.39±0.09). High genetic correlations of first lactation total milk yield (FLTMY) with first lactation standard milk yield (FLSMY, 305 days or less), FPY, and first lactation length (FLL) was seen. Likewise, genetic correlation of AFC was positive with FLTMY, FLL, first dry period (FDP), first service period (FSP), first calving interval (FCI), herd life (HL) and productive days (PD). Significant phenotypic correlation of FLTMY was observed with HL, productive life (PL), PD, total lifetime milk yield (LTMY), standard lifetime milk yield (standard LTMY). Moreover, positive genetic and phenotypic correlation of FPY was observed with HL, PL, PD, total LTMY and standard LTMY. Conclusion This study reports that AFC had positive genetic correlation with FDP, FSP, FCI, and unproductive days while, negative association of AFC was observed with FLSMY, PL, total LTMY, standard LTMY, and BE. This suggests that reduction of AFC would results in improvement of lifetime performance traits.

INTRODUCTION
India possesses the largest buffalo population (109.85 million), which constitutes approximately 56.7% of total world's buffalo population [1]. In addition, buffalo is the prime dairy animal contributing significantly to income, nutrition and employment for large rural population and hence considered as quadruple purpose animal i.e., suitable for milk, meat, draught and leather [2]. Sustainable viability of dairy enterprise depends on both production and reproduction potential of any livestock species, including buffaloes; however, livestock stake-holders neglect the fertility traits when compared to production traits [3,4]. In tropics, it has been well documented that buffaloes can efficiently utilize the roughages and crop by-products to produce high quality milk with high fat percentage and are resistant to many diseases [5]. Genetic make-up of animals governs performance of dairy farm and the knowledge of genetic and phenotypic parameters is of prime importance for designing suitable breeding policies to establish a genetically improved herd [6,7].
Selection on the basis of lifetime performance is not practically feasible due to long generation interval, so it is desirable to select the animals based on the performance of earlier lactations, rather than traits expressed later in life [8]. Likewise, suitability of breeding animals, including buffaloes, in an organized herd is primarily determined by its productive and reproductive efficiency [9]. First lactation traits are representative of subsequent lactation performance as these are highly inter-correlated both in cattle [10,11] and buffaloes [7,12]. Highly heritable traits can be used with great efficiency for genetic improvement through progeny testing as well as other genetic improvement programs. Thus, selection of animals based on the performance of the economically important traits having high heritability needs to be considered for overall improvement in lifetime productivity through correlated response [7,12].
Age at first calving is a prime reproductive trait affecting the herd's productivity as well as profitability, through direct cost of rearing bovine heifers including buffaloes [13,11] and for its effect on future performance of the animal [10]. Shorter AFC prolongs the productive life (PL) in cattle [14] and buffalo [15]. Thus, dairyman would have a considerable interest in the relationship of AFC with lifetime production and other lifetime traits, as well. Furthermore, positive association between AFC and subsequent performance was observed suggesting the potential benefits of earlier calving in buffaloes [9,6]. Attempts to reduce the AFC helps in reducing production cost and increases the profitability of dairy enterprise [17]. Understanding the production and reproduction potential along with their inter-relationship is needed to formulate strategies in breeding programs in domestic animals, including buffalo [7,18,19]. Though attempts have been made to estimate the heritability, genetic and phenotypic correlations of the economic traits in buffalo [7,12,20]. Further scope exist to analyse the inter-relationship of production and reproduction traits in buffaloes. Considering this aspect, the present study was designed to test the hypothesis of association between AFC, first lactation traits and lifetime productivity in Murrah buffaloes in organized herd.

Collection of data
Data on first lactation and life time performance of 679 Murrah buffaloes at ICAR-Central Institute for Research on Buffaloes, Hisar that remained in the herd for at least three lactations were collected for the period from 1983 to 2017. The data was analysed to calculate genetic and phenotypic parameters and the data for all the studied animals were recorded up to the date of death or culling. Animals possessing records of minimum lactation length >150 days and total milk production >1,000 kg for the first lactation were considered for the present study. Further, animals with incomplete and abnormal records due to abortion, still birth, chronic illness etc. were excluded from analyses.
In this study, herd life (HL) was considered as duration from birth to disposal of animal and PL was defined as total days from date of first calving to date of start of last dry or date of disposal if animal in lactation. Likewise, the parameter productive days (PD) was denoted by the sum of the number of days in milk in different lactations in the same herd and unproductive days (UD) was defined by the sum of dry periods in different lactations in the same herd. In this study, lifetime milk yield was defined as total amount of milk produced by a buffalo from the initiation of first lactation till the last day in milk in the herd. It was considered for the animals that remained in the herd for at least three lactations. Milk yield per day of PL was measured as lifetime milk yield divided by the PL and milk yield per day of PD is the ratio of lifetime milk yield and PD. Likewise, milk yield per day of HL was the ratio between lifetime milk yield and HL.
In this investigation, the traits analysed were AFC in months, first lactation total milk yield (FLTMY) in kg, first lactation standard (305 days or less) milk yield (FLSMY) in kg, first peak milk yield (FPY) in kg/d, first lactation length (FLL) in days, first dry period (FDP) in days, first service period (FSP) in days and first calving interval (FCI) in days and lifetime performance traits such as HL in days, PL in days, PD in days, UD in days, breeding efficiency (BE) in %, total lifetime milk yield (total LTMY) in kg, standard lifetime milk yield (standard LTMY) in kg, milk yield per day of PL (MY/PL) in kg/d, milk yield per day of productive days (MY/PD)in kg/d, milk yield per day of herd life (MY/HL) in kg/d. The present research was approved by the Institute Animals Ethics Committee (IAEC).

Statistical analyses
Data were analysed by least squares analysis model [21] using IBM SPSS Statistics 20.0 software to identify the significant fixed effects to be included in the model and determine associations between AFC, first lactation traits and lifetime productivity.
The least squares model for AFC included season of birth (4 levels) and period of birth (6 levels) as fixed effects. Fixed effects for first lactation traits were AFC (6 levels), season of first calving (4 levels), Period of first calving (6 levels); whereas for lifetime traits the fixed effects in least squares model were AFC (6 levels), season of first calving (4 levels), period of first calving (6 levels) and lactation completed (5 levels). The statistical significance was tested at 5% level and only the significant effects were included in the model that was subsequently used for analysis of genetic parameters.
The estimates of (co)variance components and genetic parameters were obtained via restricted maximum likelihood www.animbiosci.org 1153 (REML) using Wombat software [22]. Convergence was assumed if difference in log likelihood function between consecutive iterations was lower than 5×10 -4 . The univariate animal model was used in the study as follows: where y is the vector of records for different traits (FLTMY, total LTMY etc.); X is the incidence matrices assigning observations to fixed effects; β is the vector of fixed effects; Z is the incidence matrices assigning observations to random effects; u is the vector of random animal effects; e is the vector of random residual effects (error); further, bivariate analysis was performed between each pair of the first lactation and lifetime traits to estimate the genetic and phenotypic correlations.

Heritability estimates for first lactation and lifetime traits
The heritability for first lactation production and reproduction traits is presented in Table 1 Table 2).

Traits HL (d) PL (d) PD (d) UD (d) BE (%)
The genetic correlation between first lactation production and reproduction traits for Murrah buffaloes is presented in Table 3 Estimates of genetic correlation between first lactation performance and lifetime traits: The genetic correlation of first lactation production and reproduction traits with lifetime traits of Murrah buffaloes is shown in Table 5

Estimates of phenotypic correlation
Estimates of phenotypic correlation for first lactation performance traits: Phenotypic correlation for first lactation production and reproduction performance traits in Murrah buffaloes is shown in Table 1 and 7. The phenotypic correlation of production traits in Murrah buffalo was high and positive for FLTMY with FLSMY, whereas medium and positive with FPY and moderate and positive with FLL ( Table 1). The correlation of FLSMY was high and positive with FPY (0.526±0.031) and FLL (0.247±0.033) in Murrah buffalo. Phenotypic association of FDP was high and positive with FCI (0.743±0.019) and FSP (0.735±0.019) and also correlation between FSP and FCI (0.992±0.003) was observed to be high and positive.

Traits AFC (mo) FDP (d) FSP (d) FCI (d)
HL (  In this study, the phenotypic correlation between production and reproduction traits of first lactation in Murrah is shown in Table 7. FLTMY was associated positively with AFC, FSP, FCI; but negatively with FDP. Phenotypic correlation of FLSMY was high and negative with FDP. Likewise, FPY was negatively correlated with AFC; and FLL was positively correlated with AFC, FSP, and FCI. Estimates of phenotypic correlation between lifetime traits under study: Phenotypic correlation estimates among lifetime performance traits is presented in Table 2  Estimates of phenotypic correlation between first lactation performance and lifetime traits of Murrah buffaloes: Phenotypic correlation between first lactation performance and lifetime traits is shown in Table 9  positively correlated with PL, UD, and PD and negatively correlated with BE, MY/PL, MY/PD (Table 10).

DISCUSSION
For dairy enterprise, production as well as reproduction parameters are pivotal for a profitability and sustainability. In this study, an attempt was made for estimating the association between AFC, first lactation traits and lifetime productivity in Murrah buffaloes in organized herd. Heritability estimates of the traits are useful for prediction of genetic response as well as the accuracy of selection [23]. In the present investigation, lower heritability estimates of production traits, FLTMY, FLSMY, FLL, FDP, FSP, FCI, total LTMY, LTMY, MY/PL, HL, MY/HL, and PL was in agreement with earlier reports [24][25][26][27] in buffaloes corroborating that these parameters, with low additive genetic variance, are less governed by genetic influences and henceforth, can be improved through better management. In buffaloes, lower to higher heritability for milk yield have been reported in different buffalo breeds [7,[28][29][30][31] which suggests there is sufficient additive genetic variation to respond to selective breeding. These differences are probably caused by differences in locations and difference in genetic-makeup of the buffalo herd under study. Moreover, high heritability of trait such as FPY suggests that they can improve through a genetic improvement programme such as progeny testing. The moderate heritability of reproductive traits i.e., AFC deduced in this study is supported by earlier workers [7,32,33], in buffaloes, but lower heritability was observed in Murrah [34], Iranian [7], Mehsana buffaloes [23] along with Holstein Friesian cows [35]. The heritability for AFC indicates that this trait may respond reasonably well to selective breeding. A lower AFC is important because females that begin their reproductive life early can reduce production costs by increasing herd productivity. In one study [30], a lower estimate of 0.07 was obtained. In contrast, higher heritability estimates of AFC have been reported [36,37]. However, AFC is strongly influenced by the reproductive management of the herd and the lower heritability for reproductive traits except AFC in buffaloes indicate that much variation is attributed to the environmental factors and intervention through management is needed for improvement for this trait. These differences from the present investigation can be attributed to non-genetic factors environmental, breed, management, season, parity gradients used as well as the estimation methodology, which is a determinant factor in the reaction norm results [38].
With respect to genetic correlation among the first lactation performance traits, higher genetic correlation of FLTMY with FLSMY, FPY, FLL, and FLSMY with FPY and among other early performance traits hints that genes governing these traits might be common which needs to be studied in future [25]. Moreover, as FPY showed higher heritability, thus selection based on first peak yield might result in genetic improvement of other traits having high and positive genetic correlations with FPY, through correlated response [33]. The high genetic correlation between milk yield traits (FLSMY, FPY, and FLL) reported in this study implies that common genetic and physiological phenomenon affect the milk production trait. Also, a positive and high genetic correlation between the milk traits suggests the cumulative improvement of these traits, if used during selection [39].
Highly positive genetic correlation of FDP with FSP and FCI as well as FSP with FCI shows the increase in FDP and FCI with increase in FSP, since gestation period and lactation length tend to vary less in any species including buffaloes [25]. In contrast, lower genetic correlations of FPY with performance traits FSP and FCI have been reported in buffaloes [36,40]. Lower or negative genetic correlations between AFC with production traits i.e., FLTMY, FLSMY, FPY, and FLL are in agreement with earlier observations in buffaloes [7,33,41]. As compared to this study, varied estimates of genetic correlation between AFC and LL, ranging from positive to negative have been reported by different workers in buffaloes [36,42]. Likewise, positive genetic correlation between AFC and CI is supported by [30]; but negative or absence of genetic correlations between AFC and CI in different breed of buffaloes have been observed [7,37]. These varied results can be attributed to different non-genetic parameters in addition to breed difference. In buffalo, the correlations (genetic and phenotypic) between MY and LL were moderate (from 0.47 to 0.52). Moderate to high genetic correlations (0.89 and 0.72) between these traits (production traits and LL) obtained in this study is constant with earlier reports [33,43]. These results corroborate that selection for milk yield traits could affect the lactation length confirming the commonness of genes influencing the traits which can be preferred for positive genetic gain. Likewise, higher genetic correlations between lifetime traits. HL, PL, PD, total LTMY, and standard LTMY as reported earlier in buffaloes [8,27] hints the selection of animals on the basis of these different lifetime traits can be advocated for genetic improvement in buffaloes. Genetic correlation between first lactation performance and lifetime traits indicates that these traits might be controlled by similar cellular pathways as well as genes [34,44,45]. Due to positive genetic correlation for traits, selection for improvement in one trait will result in improvement in the other trait as a correlated response. Similar to our results, negative genetic correlation between AFC and PL in Murrah buffaloes was observed [46]. In accordance with our finding, positive genetic correlations of AFC with HL; FLTMY with HL, PL and total LTMY; FPY with HL, PL, and total LTMY was noticed [8,27]. AFC can be reduced through improved management practices i.e., nutritional management, health management, effective oestrus detection with insemination at the right time. Reduction in AFC to a definite level is desirable as it enhances the lifetime milk production traits total LTMY, HL, PL, and PD in buffaloes [9]. Furthermore, decrease in AFC decreases the cost of animal raising till the PL thereby increasing the annual genetic gain by decreasing the generation interval [10]. Regarding phenotypic correlation for first lactation performance traits, positive correlation between FPY and FLSMY shows the added weight given during selection as reported earlier in buffaloes [25]. Contrasting observation had been documented between production and reproduction traits in cattle [47] and buffalo [3,12,19] due to the genetic antagonism [48]. In cattle, several authors have reported improvement in milk production with improvement in reproduction in cattle [49][50][51], Brazilian Murrah [30,33,41].
Contrarily in buffaloes, negative association of FLTMY with FLL and FDP [25] as well as negative correlation between AFC and FSP with FLTMY hinting the negative effect of milk production on reproduction [52].
About lifetime traits, positive phenotypic correlations of total LTMY with lifetime performance traits HL, PL, MY/PL, and MY/HL were in agreement with previous reports in buffaloes [8,27,54]. Similarly, positive phenotypic correlations of production (FLTMY) and reproduction performance (AFC) and lifetime traits (HL, PL, total LTMY, MY/HL, MY/ PL, and UD) in buffaloes were in agreement with [8] and [27]. However, [55] reported that the herd life was not related to AFC in cattle breeds. On the contrary, negative phenotypic correlation (-0.08) between the two traits was reported in Murrah buffaloes [46] which was in agreement with our study. These variations in the outcome can be attributed to the management, environment diversity and genetic factors controlling these traits [56,57].

CONCLUSION
In this study, heritability estimates were high, moderate and medium for traits FPY, AFC, and BE, respectively. Age at first calving had positive genetic correlation with FDP, FSP, FCI, HL, and UD while, it was negatively correlated with FLSMY, PL, total LTMY, standard LTMY and BE. A younger AFC is beneficial in that it can potentially lead to a reduction in rearing costs as well as an earlier return on investment. Hence our attempts should be made towards adoption of better management practices for proper growth and development of young heifers so that they conceive and first calve earlier. Genetic and phenotypic correlation of FPY was positive with FLTMY, FLSMY, FLL, HL, PL, PD, total LTMY, and standard LTMY. The selection of animals based on FPY should be considered because FPY had high heritability and is expressed earlier in the life, so choosing animals on the basis of peak yield may lead to improvement in other lifetime performance traits such as HL, PL, PD, total LTMY, and standard LTMY along with peak yield of the animal as a correlated response. The outcome of this investigation can be effectively utilized for future selection and breeding programs in buffalo species through considering high heritable and correlated traits of economic importance.

AUTHOR CONTRIBUTIONS
TP and AB designed the study; BYC performed the statistical analysis; CA, TP, AB, JA interpreted the results; JA and TP drafted and edited the final manuscript.


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Safety, regulatory, and environmental issues related to breeding and international trade of edible insects in Africa

Insect breeding or farming for food and feed is an emerging enterprise that can address the ever-growing demand for protein and curb high unemployment rates in Africa and beyond. However, for the sector to prosper, its value chain needs to be regulated to ensure sustainability and safety for consumers and the environment. Although a few African countries, such as Malawi, Tanzania, Kenya and Uganda, have promulgated standards on the use of insects as food and feed, greater efforts are needed in other countries, and relevant policies governing the sector need to be formulated. All over the globe, attention to the regulation of the edible insect sector is peaking, and more investment in the industry is foreseen. Safety issues such as the identity of insects to be reared, substrate quality and traceability imposed by importing countries will be critical for expansion of the sector. This paper analyses safety, regulatory, and environmental issues related to breeding and international trade of edible insects in Africa and provides case studies and recommendations for sustainable use of insects for food and feed.

Introduction
Eating insects is a part of local culture for at least 2 billion people in about 113 countries (1)(2)(3)(4), and Africa accounts for over 120 million insect eaters (5). Of the 2111 edible insect species known globally (6), around 500 species have been reported in sub-Saharan Africa (7, 8). In Africa, edible insects are mainly collected from the wild for household consumption and informal trade (9-12). Wild harvesting is unsustainable due to the seasonal availability of insects, habitat change and overexploitation (9). Eating or feeding insects to animals is recommended because of their unique nutritional profile, which is 41_1_10_Niassy -pre-print 3/34 equals or exceeds that of conventional foods (13). Insects are an excellent source of protein, fatty acids, vitamins and minerals (14).
Besides traditional entomophagy (the practice of eating insects) in some parts of the world, increasing demand for alternative sources of nutrients for humans and animals has fuelled the need to farm edible insects (2, 5, 15).
Farming edible insects ensures continuous supply and offers alternative and cheaper protein sources compared to soy and fish meals (16,17). Adopting cost-effective technologies for rearing, harvesting, handling, processing, added value, and packaging is a significant game-changer to ensure constant availability of insect proteins and enhance profit margins for farmers and other stakeholders (7, 8). Insects grow faster with high feed conversion efficiency and have less requirements for space and water than many conventional feed species. The carbon footprint of insect farming is much lower than conventional farming (2, 4,18). Insect farming is gender-friendly (15,19,20) and currently, there are nearly 1000 edible insect farms in Africa, with the industry projected to be worth up to US$8 billion by 2030 (5). A recent World Bank report estimates that black soldier fly farming alone has the potential to replace 60 million tons of traditional feed production in Africa annually, leading to 200 million tons of recycled crop waste, 60 million tons of organic fertilizer production, besides creating 15 million jobs (5). Therefore, the use of insects as food and feed may help attain some UN Sustainable Development Goals and the aspirations of the African Union Agenda 2063 (21).
Although insect farming is considered an emerging enterprise in Africa (5, 15,22), critical issues in the production chain need undivided attention if the sector is to prosper. Key among these is safety, and the most debated issues are food hazards commonly associated with edible insects and their by-products. These issues may be related to how the insects have been reared, euthanised, handled, processed and packaged.
Biological agents and chemical contaminants are some major health hazards associated with edible insects (2). Safety issues around insects for food and feed cut across environmental, health, agriculture and trade.
41_1_10_Niassy -pre-print 4/34 Food safety regulations on edible insects are not fully realised in Africa due to a wide range of challenges (23). These challenges include: 1) lack of awareness of food safety on socio-economic matters; 2) lack of established hazard and critical point checklists/systems; 3) limited data and information on health burden, especially food-borne diseases; 4) limited compliance with international agreements on food safety and quality standards; 5) limited enforcement of local, regional and international standards and global best practices; 6) limited resources to support scientific risk analysis and upgrading of food safety regulatory systems; and 7) poor food supply chains and traceability system.
Several authors acknowledged the lack of regulatory frameworks specific to insect farming and their use as food and feed in many countries (18,24,25). There is also a lack of harmonisation to improve policy and strengthen the trading environment, especially market linkages, to reduce constraints along the insect-based value chain (25).
Several regulatory bodies are nominally in charge; however, procedures and the motivation behind these safety measures on food are often unclear, overlooked or fail to capture specific matters that concern the farming of insects as "mini-livestock". The lack of understanding of these regulatory and legislative measures vis-a-vis the diversity of edible insects (which belong to several insect orders with very different lifestyles) hampers the sector. The objective of the present paper is to analyse the regulatory elements that govern safety issues in edible insect farming for national and international trade and provide an overview of legal procedure flow among regions and countries in Africa. The production chain and safety and regulation in relation to the environment and trade are summarized in Fig. 1. resistant bacteria are also of major concern. High levels of microbial pathogens will raise health concerns and consequently affect the consumption and sale of edible insects and insect-based products at a local, national, regional and international scale.  (2).

Pesticides residues and toxic, trace metals
Pesticides residues and toxic metals in reared insects can be associated with particular insect species, their source before being reared, sources and type of substrates used and contamination of the rearing unit (2, 39

Other substances
According to FAO (2), edible insects and insect products may contain, accumulate or release noxious substances. These substances include bioaccumulated organic compounds (organic contaminants, plasticisers, and halogenated flame retardants), dioxin-like polychlorinated biphenyls (PCB), spiked levels of mineral oil hydrocarbons and histamines. Other undesirable contaminants such as antimicrobials, allergens and inherent anti-nutritional substances (phytic acids, tannins, saponins, phenolics, thiaminase, cyanogenic glycosides and oxalates) may accumulate in edible insect products during production and processing (2). Poorly processed and packaged edible insect products may contain undesirable objects and hard parts of the insects such as wings, rostrum, stings and spines.

Allergies arising from eating insect-based products
Insects and crustaceans are related members of the phylum Arthropoda.
Since allergic reactions to crustaceans are potentially severe, a cross-41_1_10_Niassy -pre-print 9/34 reactivity and co-sensitisation of the immune system between edible insects, crustaceans and mites pose a potential health concern (43).
Indeed, cases of cross-reactivity/co-sensitisation between edible insects and crustaceans leading to food allergy have been clinically relevant with an unknown underlying molecular mechanism attributed to allergens, specifically arginine kinase (44 and references therein). Food allergy due to consumption of edible insects has been described for silkworms, mealworms, caterpillars, locusts, grasshoppers, cicadas, bees and sago worms (45). There are opportunities for further research on allergies due to eating insects, including diagnostic allergy tests that could lead to the identification of vaccines.

National, regional and international regulatory frameworks for edible insects
As insect farming is gaining prominence, questions on safety are increasingly important. Countries that are known to practise entomophagy lack regulatory policies that would potentially benefit the insects for food and feed sector. These countries rely on a precautionary approach or the World Health Organization (WHO) and Food and Agricultural Organization (FAO) information on insect-based diets (18). Most studies have clearly shown that there are more stringent rules on the use of insects as "novel food and feed ingredients" in countries with no history of entomophagy but have food policies that prioritise food safety (18,46). Country specific regulations have also been formulated in some countries (47). can be considered to be either in "traditional" or "transitioning" stages towards "modernising" food systems. While African countries are in the "transitioning" stages of recognising edible insects as "foodstuffs" or "animal feed", it is expected that most existing food laws and policies would apply to edible insects as well.
Edible insects are not listed in the Codex Alimentarius, which is considered a global problem (53 However, in the case of EU standards, insect species used for food and feed must be reared on a substrate that fulfils certain prescribed safety conditions for insect production (53,56,57). According to the EU, insects used as food and feed should not be reared on waste streams (2, 57); however, insects meant for biomass conversion for economic gains can be reared on waste streams (2). Though EU restrictions are designed to eliminate the risk of prion contamination of the edible insects, they limit their use to close nutrient loops, this is also the case for Africa.

South Africa
In South Africa, four government departments oversee food safety and quality legislation (Table I) (Table II).      Regulatory procedure of the insect production chain and the emergencies in relation to safety in Kenya