Coffee By-Products and Chitosan for Preventing Contamination for Botrytis sp. and Rhizopus sp. in Blueberry Commercialization

Abstract

In blueberry storage, non-biodegradable synthetic plastic packaging is used for commercializing this product. The fungi Botrytis sp. and Rhizopus sp. can cause significant losses in postharvest blueberry commercialization. Consequently, the formulations of degradable polymeric based on polylactic acid (PLA)/poly(butylene adipate-co-terephthalate) (PBAT) 60/40 (PP) with coffee parchment (CP), green coffee bean oil (GCBO), chitosan solution (Ch), chitosan nanoparticles (ChNp), and nanostructured coating (NC) were used to develop biodegradable polymer matrix (PM). Caffeine and hexadecanoic acid were identified as principal compounds in GCBO, and the principal compounds in CP were flavonoids, terpenes, and lignans. The 100% mycelial growth inhibition to Botrytis sp. and Rhizopus sp. was observed using GCBO, Ch, ChNp, and NC in high concentrations. GCBO inhibited 100% of spore production in both fungi at all evaluated doses. In the in vivo tests, when compared to the control, the better treatments were: CP for Botrytis sp., with an incidence of 46.6% and a severity of 16%; and Ch for Rhizopus sp., with an incidence of 13.3% and a severity of 0.86%. The PM in the culture medium presented a fungistatic effect. The principal inhibition of mycelial growth (63%) on Botrytis sp. was with PLA/PBAT+NC (PP+NC), and (100%) was observed with PLA/PBAT+CP+NC (PPCP+NC), PP, and PP+NC on Rhizopus sp. Coffee by-products and PM have potential for the control of postharvest fungi in fruits and vegetables.

1. Introduction

Blueberry (Vaccinium corymbosum) is a fruit with high nutritional value, low caloric content, high antioxidant content, and anti-inflammatory properties [1]. These attributes make it an attractive product due to its versatility and benefits for human health. For these reasons, its popularity and demand have increased in the last decade, increasing production and cultivated areas in different regions of the world. Fungal diseases are the main causes of the reduction in plant productivity. The most recurrent genera are Botrytis and Rhizopus, which affect the fruit in the postharvest stage [2]. In blueberries, losses of up to 30% in total production have been recorded due to these phytopathogens [3]. Infections caused by Botrytis sp. are probably the most common worldwide, with a wide distribution and a wide range of crops [4]. The disease it causes tends to be more severe in cold and humid environments; this fungus can develop even at 0 °C. Rhizopus sp. causes soft rot in most fleshy fruit during storage and marketing; when growing conditions are favorable, it can cause large losses in a short period of time due to its rapid development [5]. Today, there are several methods for combating disease-causing agents in agriculturally important crops; however, indiscriminate pesticide use has led to the creation of new biological alternatives that help combat this problem [6]. One of these proposals is to use compounds of natural origin; several plants contain bioactive agents that act against disease development in agriculturally important crops [7]. The high production of coffee generates a large quantity of waste that has a wide application due to its physical and chemical properties, bioavailability, and potential in food production, making it useful in various fields [8].

Green coffee bean oil is obtained from unroasted beans and has several applications in both the cosmetic and food industries due to the presence of bioactive compounds, such as polyphenols, tocopherols, and phytosterols [9]. The (CP) is the covering of the bean at the parchment stage and is considered an agro-industrial waste because its use is in low demand. Recently, it has been sought to explore new applications because CP is a by-product rich in lignocellulosic matter and contains antimicrobial compounds, such as alkaloids and flavonoids, that function as a defense against coffee pathogens and pests [10]. Extracts and oils can be obtained by conventional extraction methods and solvents [11]. Gloria et al. [12] attributed the antimicrobial activity of coffee to caffeine, trigoline, and phenolic acids and their derivatives, and after the bean roasting process it was attributed to melanoids and dicarbonyl compounds. Generally, the bioactive compounds present in coffee are chlorogenic acids, quinic acid, malic acid, and caffeine [13]. Another naturally occurring compound is chitosan, which is obtained from the chitin of some mollusks [14]. It has a high impact on the use of biopolymers and green chemistry due to its high antimicrobial activity. This property depends on several factors, such as the type of pathogen, pH of the medium, and the structure and concentration of chitosan [15]. One of the main uses of chitosan is in the preparation of coatings for fruit and vegetable preservation; they help to reduce deterioration due to the presence of microorganisms and maintain fruit quality [16]. Sotelo-Boyás et al. [17] found that using chitosan nanoparticles enhanced their antimicrobial effects because the nanoparticles could more easily penetrate the wall of the microorganisms. Other polymers used to produce packaging are of synthetic origin, such as polyethylene terephthalate, polystyrene, and polypropylene, which have a negative impact on the environment and health [18]. Today, there is great interest in generating polymers of natural origin that are biodegradable and compostable [19]. Polylactic acid (PLA) is a biodegradable polymer because it is a derivative of lactic acid obtained from renewable resources, such as corn starch and sugar cane, which makes it a polymer of great importance today [20]. Poly(butylene adipate-co-terephthalate) (PBAT) is a synthetic polymer obtained from fossil-based resources; however, it has the capacity to be biodegradable and the potential to be used in various applications [21]. More knowledge of the management and handling of plastics is needed since the main problem they have faced is a lack of information on their origin and adequate treatment, either recycling or composting for their reintegration [22]. Currently, in the food industry, there is a search for incorporating bioactive compounds of natural origin to improve the organoleptic characteristics of fruit and vegetable products, extend their shelf life, and prevent their deterioration by external agents, such as microorganisms [23]. The use of chitosan as an antimicrobial has been studied previously, and it was demonstrated that, by synthesizing chitosan nanoparticles, their antimicrobial effect can be improved [24]. Recent work has been carried out on the synthesis of nanoparticles from extracts obtained from coffee waste [25].

This study aimed (i) to identify chemical compounds of GCBO and CP using chromatographic techniques to know the principal compounds in these coffee wastes; (ii) investigate the antifungal activity of individual coffee residues in vitro and in vivo against Botrytis sp. and Rhizopus sp., and be able to use them as bioactive compounds for disease control; and (iii) evaluate the antifungal in vitro effect of the incorporation of CP and GCBP in a biodegradable polymeric matrix (PM) and be able to reduce the cost of production of these polymers and generate a new ecological alternative.

2. Materials and Methods

Figure 1 shows the research methodology, supplemented in the succeeding parts of this article.

2.1. Materials

The CP and green coffee bean residue were provided by the coffee producers of Coatepec Veracruz, Mexico. The CP was cleaned and separated manually to eliminate external agents. They were then subjected to a drying process using an oven (Binder FD 115, Tuttlingen, Germany) at 60 °C for 12 h to eliminate the total content of humidity. After drying, grinding was carried out in a pulverizing mill (INMIMEX M-150, Tlaxcala, Mexico) and the dust particles that passed through a 100-mesh sieve were recovered. The powders were stored in a container until use. To obtain the oil, ground green coffee beans were used and sifted through a 60-mesh sieve. The Soxhlet extraction system used 50 g of sample placed in the cartridge and left for 6 h in the reflux system with hexane. The extracted oil was recovered and then concentrated in a rotary evaporator (BUCHI R-300, Flawil, Switzerland) to eliminate solvent residues. The sample was packaged and stored in a refrigerator.

2.2. Characterization of Coffee Oil by Gas Chromatography and Mass Spectrometry (GC-MS)

To identify compounds from green coffee beans oil using gas–mass chromatography, (Agilent Technologies 890B Gas Chromatograph Los Angeles, CA, USA), with a thermal separation probe (Agilent G4381A-TPS) and 0.6 mg of oil was placed in a 50 µL TSP microvial and analyzed. The equipment was operated under the following conditions: helium as carrier gas, an initial temperature of 80 °C, a final temperature of 300 °C, an injector temperature at 250 °C, and a run time of 45 min. The mass detector operated under the following conditions: mass range of 32–750 atomic mass units (MS), at a transfer temperature of 280 °C, and with a solvent delay of 0.2 min. The identification of the major compounds was carried out based on the spectra of the NIST library.

2.3. Characterization of CP by Liquid Chromatography and Mass Spectrometry (HPLC-MS)

To identify CP compounds, 1 g of CP was deposited in 10 mL of methanol, and 100 µL was diluted in 900 µL of distilled water and directly injected (10 µL) into a Chromatograph (HPLC) Ultimate 3000 (Dionex Corp, CA, USA) equipped with an array of diodes and a micrOTOF Q-IIanalyzer in electrospray ionization (ESI) system mode (Bruker Daltonics, Billerica, MA, USA). Components were separated using reverse-phase Acclaim 120, reverse-phase (RP)-C18 120 Å, 2.1 × 150 mm, and 3.0 μm column (Dionex, Sunnyvale, CA, USA) and held at 50 °C [26]. MzCloud, MassBank and bibliographic databases were used to identify the compounds.

2.4. Environmental Scanning Electron Microscopy (ESEM-EDS)

For elemental analysis of CP, the samples were placed on aluminum stubs with double-sided carbon conductive tape and were observed directly under an environmental scanning electron microscope (Carl Zeiss, EVO LS10, Weimar, Germany) with an acceleration voltage of 30 kV and a pressure of 90 Pa. A backscattered electron detector (NTS BSD) was used, and images were obtained in grayscale and stored in TIFF format with a resolution of 1044 × 756 pixels.

2.5. Nanoparticles and Nanostructured Coating Elaboration

The nanoparticles were prepared using the nanoprecipitation method, which consisted of using a solvent phase composed of 96% ethanol and 0.1 mL of Tween 20 for every 100 mL of ethanol. In addition, a second phase composed of a 0.05% chitosan solution (América Alimentos, Guadalajara, Mexico); 6.25 mL per 100 mL of ethanol, pH 5.6) was used. The chitosan had an intrinsic viscosity of 440.03 ± 6.92 mL∙g⁻¹, a molecular weight of 89,305.66 ± 1850.49 g∙Mol⁻¹, and a degree of deacetylation of 89.44 ± 0.31%. Nanoparticle formation and concentration were determined according to the method proposed by Istúriz-Zapata et al. [27] and were previously characterized by Istúriz-Zapata et al. [28]. To prepare the chitosan coating with the nanoparticle solution, it was mixed in a 9:1 ratio with a 1% chitosan solution (pH 5.6) and a homogenizer (Virtis, Garndier, Warminster, PA, USA) at 20,000 rpm for 20 min.

2.6. Funtional Groups of CP, GCBO, and ChNp by FTIR

The functional groups present in the CP, GCBO, and ChNp were obtained using a Confocal Micro-Raman Spectroscopy equipment coupled with Fourier Transformed Infrared (FTIR) (CRAIC Technologies, Whitehall, PA, USA).

2.7. Incorporation of Coffee Waste and NC in a Polymeric Matrix

To produce PM, mixtures of PLA and PBAT polymers were pelletized in a 60/40 ratio according to Correa-Pacheco et al. [29], who mentioned that the mixture of these polymers improved the physical characteristics, such as greater flexibility and an increase in the elongation capacity at break. In addition, 5% CP was used as it was the optimal loading point for better physical characteristics. To obtain the PM, an extrusion process was carried out using a twin-screw extruder (Process 11, Thermo Scientific TM, Waltham, MA, USA), which had eight heating zones, a die, and four feed ports. The following temperatures were used: 160, 180, 180, 180, 180, 170, 170 and 150, and the die at 160 °C. The PLA/PBAT 60/40 pellets with 5% CP were previously dried in a vacuum oven at 60 °C for 24 h to remove moisture. These were placed in the first feed port while the GCBO was incorporated into the second feed port using a peristaltic pump (MasterFlex C/L, Cole-Parmer, Vernon Hills, IL, USA). At an injection speed of 0.2 rpm, the purpose of incorporating GCBO was to take advantage of its antimicrobial and plasticizing properties that provide greater flexibility, lower rigidity, and crystallization, improving the mechanical properties of the polymer [30]. Two variables were used in the cooling bath: one was to use water and the second was to use NC to adhere to the PM.

2.8. Fungal Genera

Botrytis sp. and Rhizopus sp. were obtained from the collection of the CEPROBI-IPN Postharvest Technologies Laboratory, identified by their morphological characteristics and reactivated in potato dextrose agar culture medium. The Petri dishes were incubated at 12 °C ± 2 for 8 days for Botrytis sp. and 28 °C ± 2 for 43 h for Rhizopus sp.

2.9. Mycelial Growth Inhibition

The method reported by Velázquez Silva et al. [31] was used for the evaluation. The treatments consisted of control potato dextrose agar (PDA), CP (1, 5, and 10%), GCBO (1, 3, and 6%), NC (1, 10, and 20%), Ch (1, 10, and 20%), and ChNp (1, 10, and 20%). Then, 6 mL of each mixture was added to Petri dishes of 9 cm diameter. Once the culture medium solidified, 10 μL of a spore suspension (1 × 10⁻⁵) of Botrytis sp. and Rhizopus sp. was placed in the center of the dish and incubated under the conditions mentioned above. The radial growth of the fungus was measured using a Vernier caliper (Cienceware, CA, USA) until the control treatment covered 100% of the Petri dish. The results were reported in millimeters (mm). Six repetitions were carried out per treatment (n = 6) and, at the end of the test, the percentage of mycelial inhibition was calculated using the following equation:

%MI = [(A − B)/A] × 100,

(1)

where A is the mycelial growth of the control group and B is the mycelial growth of the pathogen in the applied treatments.

The growth rate was calculated using the equation:

Gr = [(DF−DI)/(FT−IT)],

(2)

where Gr is the growth rate, DF is the diameter of the final growth, DI is the diameter of the initial growth, FT is the final time of mycelial growth in days or hours, and IT is the start time.

For percentage data, a Bliss angular transformation was used using the following equation:

y* = arcsen√(y/100)

(3)

2.10. Spore Germination Inhibition

The mycelium was scraped with sterile water and filtered to obtain a spore suspension of each treatment. Three discs of 1 cm diameter PDA medium were placed on a sterile slide; the discs were inoculated with 20 µL of the spore suspension. Spore germination was stopped by applying 80% lactic acid at 4, 6, and 8 h, and the number of germinated spores was counted. The percentage of spore germination inhibition was calculated using the equation:

SGI (%) = [(SG Control−SG Treatment)/SG Control] × 100

(4)

where SGI is the spore germination inhibition in percentage and SG are the germinated spores.

2.11. In Vivo Evaluation

Blueberry fruit (variety Sweetest batch) collected from Zapopan, Jalisco, Mexico, was used. The fruit was disinfected with a 400 ppm sodium hypochlorite solution for 3 min and rinsed with sterile distilled water. The 1 × 10⁻⁵ spore suspension was prepared for the fungi Botrytis sp. and Rhizopus sp., and 10 µL of the suspension was placed on each fruit, previously punctured with a sterile dissection needle. The fruit was immersed for 1 min in different treatments for evaluation. For ground pure CP, a 1:1 portion of CP and sterile distilled water were used and compared with a commercial fungicide (CAPTAN 500 MezFer) at 3%. Fifteen fruits were used for each treatment, and each treatment was placed in a PET clamshell and stored at room temperature (28 ± 2 °C) under relative humidity conditions (95 ± 2%). Evaluations were made every day, and severity and incidence were determined using the equation:

%Incidence = (Number of infected fruits/Total number of fruits) × 100

(5)

Severity was determined by the percentage of fruit damage.

2.12. In Vitro Assay for the PM

The method described by Correa-Pacheco et al. [32] was used with modifications. A 5 mm circle of the PM was placed in the center of the Petri dish with PDA medium, and 10 μL of the 1 × 10⁻⁵ spore suspension of Botrytis sp. and Rhizopus sp. was added. The radial growth of the fungus was measured using a Vernier caliper (Cienceware, CA, USA) until it covered 100% of the Petri dish in the control treatment. Six repetitions were carried out per treatment (n = 6) and, at the end of the incubation time, mycelial growth was reported. The treatments evaluated in this trial were PLA/PBAT (PP), PLA/PBAT with chitosan coating (PP+NC), PLA/PBAT with CP (PPCP), PLA/PBAT with CP and chitosan coating (PPCP+NC), and Polyethylene Terephthalate (PET).

2.13. Statistical Analysis

In vitro results were analyzed by a one-way analysis of variance (ANOVA) with Tukey’s comparison of means (p < 0.05). InfoStat software version 2020 was used.

3. Results

3.1. Characterization of Green Coffee Bean Oil by GC-MS

Six compounds were identified in green coffee bean oil. Figure 2 shows the detection peaks and

Table 1. Compounds identified by GC-MS in green coffee bean oil.

Peak n°	RT	Compound	% Abundance	Area	CAS
1	14.94	Caffeine	10.159	506,873	58-08-2
2	16.00	n-Hexadecanoic acid	14.344	715,646	57-10-3
3	17.64	17-Octadecynoic acid	12.244	610,906	34450-18-5
4	22.40	1H-Indene, 2,3-dihydro-4,7-dimethyl-	19.759	985,831	6682-71-9
5	22.96	Benzimidazole, 2-methyl-1-(3-phenylpropylthio)methyl-	10.540	10.540	615-15-6
6	23.19	Norethindrone	20.9440	20.944	68-22-4

shows the following composition: 10% caffeine alkaloid, 26.5% fatty acids, 30% methylated aromatic compounds, and 20.5% hydroxy-steroid. These compounds have antioxidant and antimicrobial properties that can be widely used in the food industry.

3.2. High-Performance Liquid Chromatography (HPLC) Analysis

A total of 21 major compounds were identified in CP. Most of the identified compounds were phenolic compounds, terpenes, and lignans (Figure 3,

Table 2. Chemical composition of coffee parchment, identified by HPLC.

Peak n°	RT	Compound	Area	CAS	Formula
8	9.6	Delphinidin 3-O-galactoside	96,352	197250-28-5	C21H21O12
10	9.9	Delphinidin 3-O-arabinoside	115,798	28500-01-8	C20H19O11
11	10.7	Kaempferol 3-O-rhamnoside	216,143	482-39-3	C21H19O10
12	11.9	Isorhamnetin 3-O-galactoside	397,750	5041-82-7	C22H22O12
13	13	Quercetin 3-O-acetyl-rhamnoside	675,166	Not available	C23H22O12
14	14	Theaflavin	2,433,133	4670-05-7	C29H24O12
15	15.0	Andrographolide	1,412,321	5508-58-7	C20H30O5
20	15.4	Kaempferol	9,936,525	520-18-3	C15H10O6
24	16.3	Kaempferol 3-O-(6″-acetyl-galactoside) 7-O-rhamnoside	658,288	124097-45-6	C29H32O16
28	17.3	Malvidin 3-O-(6″-p-coumaroyl-glucoside)	9,548,757	158189-28-7	C32H31O14
33	18.4	Biochanin A	1,985,709	491-80-5	C16H12O5
37	19.5	Ligstroside	958,156	35897-92-8	C25H32O12
42	20.7	3-Hydroxyphloretin 2′-O-xylosyl-glucoside	1,290,713	Not available	C26H32O15
48	22.6	Resveratrol 3-O-glucoside	1,275,343	38963-95-0	C20H22O8
51	23.7	p-HPEA-EDA	8,929,194	151194-92-2	C17H20O5
55	24.4	Secoisolariciresinol-sesquilignan	446,635	Not available	C30H38O10
56	24.5	Isorhamnetin 3-O-rutinoside	801,069	Not available	C22H22O11
57	24.6	Luteolin 7-O-glucuronide	462,237	29741-10-4	C21H18O12
59	25.4	2-[4-(Diethylamino)-2-hydroxybenzoyl]benzoic acid	8,324,925	5809-23-4	C18H19NO4
60	25.5	2-S-Glutathionyl caftaric acid	21,393,122	Not available	C23H27N3O15S
61	25.7	Dehydroeburicoic acid	48,266,948	6879-05-6	C31H48O3
61	25.9	Dihydromyricetin 3-O-rhamnoside	4,582,788	Not available	C21H22O12
64	26.4	Cyclolariciresinol	390,934	548-29-8	C20H24O6
71	29.2	Artemisinin	500,292	63968-64-9	C15H22O5

), such as the anthocyanins delphinidin 3-O-galactoside and delphinidin 3-O-arabinoside, the flavonols quercetin 3-O-acetyl-rhamnoside and kaempferol, a biflavonid theaflavin, diterpenoid andrographolide, isoflavone biochanin A, secoiridoid glycoside ligstroside, stilbenoid resveratrol 3-O-glucoside, phenols and alcohol p-HPEA-EDA, lignans secoisolariciresinol-sesquilignan and cyclolariciresinol, disaccharide derivative isorhamnetin 3-O-rutinoside, trihydroxyflavone luteolin 7-O-glucuronide, glycosidal flavonoid dihydromyricetin 3-O-rhamnoside, and sesquiterpene lactone artemisinin (database consulted: PubChem).

3.3. Elemental Analysis of Coffee Parchment

The morphology of the ground and sifted CP is observed in microscopies, with heterogeneous sizes and shapes, obtaining elongated shapes larger than 20 µm (Figure 4).

In the elemental analysis by EDS, oxygen and carbon were observed as major elements within CP (42 and 57%, respectively), and 1% corresponds to minor elements such as calcium (0.08%), silicon (0.05%), sulfur (0.02%), calcium (0.13%), iron (0.13%), potassium (0.01%), and fluorine (0.11%). This study confirms that the CP sample did not contain organic pollutants within its composition (Figure 5).

3.4. Identification of Funtional Groups of CP, GCBO, and ChNp by FTIR

The FTIR spectra shows the functional groups of the CP, GCBO, and ChNp in Figure 6. In the CP, it shows the hydroxyl group (O-H) between 3400–3500 cm⁻¹, that means the stretch of absorbed cellulose bond, between 2800–3000 cm⁻¹ it shows the stretch of the C-H bons, present in hemicellulose, and between 1000–1200 cm⁻¹ it shows the absorption of C-O-C bonds, part of the vibrations of cellulose pyranose ring vibration [33]. In the GCBO, the peaks between 3000 cm⁻¹ represent the symmetric vibration of C-H stretching in aromatic rings, and the peaks between 2800–2900 cm⁻¹ show the methylene group (-CH₂) and the methyl groups (-CH₃); these groups are presents in lipids [34]. The peaks between 1500–1400 cm-1 represent the chlorogenic acid [25]. ChNp is associated with the hydroxyl group (-OH) between 3400–3600 cm⁻¹ and the carbonyl group of the amino group (-NH₂) located between 1600–1500 cm⁻¹ [35].

3.5. In Vitro Effect of Coffee Residues, Chitosan, and Chitosan Nanoparticles on Mycelial Growth and Spore Germination Inhibition of Botrytis sp. and Rhizopus sp.

In the case of Botrytis sp., a mycelial growth rate (MGR) of 7.25 mm/day was observed in the control (PDA), similar to the treatments of CP 1% and CP 5% with an MGR of 7.19 and 6.55 mm/day, respectively. These treatments with CP presented a stimulatory effect on mycelial growth; with the exception of CP at 10%, the inhibitory effect was (51.98%). In contrast to GCBO in the three concentrations, which presented 100% inhibition. Additionally, the treatments with NC, Ch, and ChNp at concentrations of 10 and 20% showed a greater inhibitory effect (100%), as shown in

Table 3. In vitro antifungal effect of coffee residues, chitosan, and chitosan nanoparticles on Botrytis sp.

Treatment	Grow Rate (mm/day)	Mycelial Growth on the Last Day	Inhibition of Mycelial Growth (%)	Inhibition of Spore Germination (%)
Control	7.25	50 ± 0 d	0 a	0
CP 1%	7.19	48.14 ± 1.11 d	3.72 bc	−7.98
CP 5%	6.55	44.31 ± 4.22 c	11.38 c	4.19
CP 10%	3.16	24.01 ± 3.42 b	51.98 d	NSF
GCBO 1%	NG	0 a	100 f	NSF
GCBO 3%	NG	0 a	100 f	NSF
GCBO 6%	NG	0 a	100 f	NSF
NC 1%	7.09	47.54 ± 1.20 d	4.92 bc	−3.39
NC 10%	NG	0 a	100 f	NSF
NC 20%	NG	0 a	100 f	NSF
Ch 1%	7.37	49.43 ± 0.45 d	1.14 ab	−3.39
Ch 10%	NG	0 a	100 f	NSF
Ch 20%	NG	0 a	100 f	NSF
ChNp 1%	7.18	48.08 ± 1.640 d	3.84 b	−8.98
ChNp 10%	NG	0 a	100 f	NSF
ChNp 20%	NG	0 a	100 f	NSF

CP = coffee parchment; GCBO = green coffee bean oil; NC = nanostructured coating; Ch = chitosan solution; ChNp = chitosan nanoparticles. One-way ANOVA, Tukey α = 0.05; F = 2164.85; gl = 74; standard error = 1.4207; p < 0.0001. Different letters indicate significant differences, NSF = no spore formation, NG = no growth.

and Figure 7A. Most treatments inhibited the formation spores, with the exception of the 1% CP, 1% NC, 1% Ch, and 1% ChNp treatments, which presented higher percentages of SGI compared to the control; only the CP 5% treatment presented a slightly positive percentage of germination inhibition of Botrytis spores compared to the control.

In Rhizopus sp., an MGR of 1.26 mm/h was observed in the control (PDA), while in the CP at the different concentrations, a higher rate was observed due to the stimulation of mycelial growth. In the case of GCBO, a decrease in the MGR was observed (0.66 for 1%, 0.54 for 3%, and 0.83 for 6%) and MGI of 43.38 to 31.5%. For NC and Ch at the highest dose of 20%, the inhibition mycelial growth was 100%; however, the 1% dose presented a stimulation with an MGR of 1.28 mm/hour. In ChNp, the lowest dose of 1% presented an MGR of 1.24 mm/hour, but the doses of 10 and 20% did not show mycelial growth. These data can be compared with the inhibition percentages shown in

Table 4. In vitro antifungal effect of coffee residues, chitosan, and chitosan nanoparticles on Rhizopus sp.

Treatment	Grow Rate (mm/Day)	Mycelial Growth on the Last Day	Inhibition of Mycelial Growth (%)	Inhibition of Spore Germination (%)
Control	1.26	50 ± 0 d	0 a	0 a
CP 1%	1.28	50 ± 0 d	0 a	12.93 ef
CP 5%	1.28	50 ± 0 d	0 a	17.86 ef
CP 10%	1.28	50 ± 0 d	0 a	16.25 f
GCBO 1%	0.66	28.31 ± 7.41 b	43.38 de	NSF
GCBO 3%	0.54	24.20 ± 2.01 b	51.6 e	NSF
GCBO 6%	0.83	34.25 ± 1.17 c	31.5 d	NSF
NC 1%	1.24	48.55 ± 1.19 d	2.9 b	4.72 bcd
NC 10%	0.54	24.14 ± 6.03 b	51.72 e	1.92 b
NC 20%	NG	0 a	100 f	NSP
Ch 1%	1.28	50 ± 0 d	0 a	8.21 cde
Ch 10%	1.14	45.02 ± 0.69 d	9.96 c	3.32 def
Ch 20%	NG	0 a	100 f	14.51 bc
ChNp 1%	1.24	48.63 ± 1.28 d	2.74 b	NSF
ChNp 10%	NG	0 a	100 f	NSF
ChNp 20%	NG	0 a	100 f	NSF

CP = coffee parchment; GCBO = green coffee bean oil; NC = nanostructured coating; Ch = chitosan solution; ChNp = chitosan nanoparticles. One-way ANOVA, Tukey α = 0.05; F = 379.76; gl = 74; standard error = 6.3158; p < 0.0001. Different letters indicate significant differences. NSF = no spore formation, NG = no growth.

and Figure 7B. The treatments that presented a 100% inhibition of the mycelial growth of Rhizopus sp. were 20% NC, 20% Ch, and 10 and 20% ChNp, while treatments with CP did not inhibit mycelial growth. Coffee oil and chitosan nanoparticles at all concentrations inhibited spore formation. The percent inhibition of spore germination presented values from 1.92% to 17.86%, and presented significant differences (p < 0.0001) between the treatments of CP, NC, and Ch. In the germination inhibition of Rhizopus spores, the CP treatments presented the highest percentages of SGI, together with 20% Ch; however, all treatments with GCBO and ChNp presented no spore formation. High concentrations prevented mycelium development, but spores were not formed at low concentrations despite the presence of mycelium.

3.6. In Vitro Assay of Coffee Residues and Incubation Times on Mycelial Growth and Spore Germination Inhibition of Botrytis sp. and Rhizopus sp.

There were no differences in mycelial growth of Botrytis sp. between incubation times or between treatments at different concentrations. The 10% CP treatment showed an initial growth of 5 mm to 24 mm at 8 days compared to the 50 mm control (Figure 8a). In Rhizopus sp., the 1% GCBO, 3% GCBO, 6% GCBO, 10% NC, and 10% CP treatments showed the lowest mycelial growth with significant differences compared to the control (p < 0.001). (Figure 8b).

In the inhibition of spore germination of Botrytis sp. and Rhizopus sp. with respect to time, no significant differences were observed (p < 0.001) between the treatments, except for the treatment with 10% NC at 4 and 6 h on Rhizopus sp., as shown in Figure 9.

3.7. In Vivo Effect

Variations in incidence and severity were observed in blueberry fruits. The in vivo test for Botrytis sp. showed the lowest incidence after 10 days of evaluation (40%) with the commercial fungicide; it was more effective than the other treatments and the control (66.66%). The CP and NC treatments presented an incidence of 46.66%. In terms of severity, the control treatment showed the highest values with 34%, while the NC treatments (17.73%) and CP (16.66%) were more effective in reducing fruit damage (Figure 10 and Figure 12(1-a,2-a)).

For Rhizopus sp., the in vivo test was 4 days (Figure 11 and Figure 12(1-b,2-b). The incidence in the control was the most susceptible with 100%, while the lowest incidence was observed in Ch treatment (13.33%). The severity in the control group reached 100%, while the Ch treatment was the most effective with a severity of 0.86%. All treatments had the lowest incidence and severity on blueberry.

3.8. In Vitro Assay of Coffee Residues Incorporated into the PM

The results are shown in

Table 5. In vitro effect of PM on Botrytis sp. and Rhizopus sp.

	Botrytis sp.		Rhizopus sp.
Treatment	Mycelial Growth on the Last Day (mm)	Inhibition of Mycelial Growth (%)	Mycelial Growth on the Last Day (mm)	Inhibition of Mycelial Growth (%)
Control	50 ± 0	0 a	50 ± 0 b	0 a
PET	44.16 ± 16	11.68 b	42.86 ± 3.68 b	14.28 b
PPCP	40.61 ± 2.08	18.78 b	42.11 ± 8.09 b	15.78 b
PPCP+NC	40.60 ± 4.99	18.8 b	0 a	100 c
PP	27.98 ± 2.62	44.04 c	0 a	100 c
PP+NC	18.48 ± 5.87	63.04 d	0 a	100 c

Control = PDA, PET = Polyethylene Terephthalate, PPCP = PLA/PBAT with CP without NC, PPCP+NC = PLA/PBAT with CP and NC, PP = PLA/PBAT without NC and PP+NC = PLA/PBAT with NC.

. A fungistatic effect was obtained in all treatments, with significant differences (p < 0.0001). The PP+NC treatment was the most effective with a mycelial growth inhibition of 63.04%, while the least effective was PET with an inhibition of 11.68% on Botrytis sp. compared to the control. In Rhizopus sp., a fungistatic effect was also obtained. Compared to the control, mycelial growth was inhibited by 14.28% with PET treatments and 15.78% with PPCP treatments. The PPCP+NC, PP, and PP+NC treatments showed 100% inhibition mycelial growth during the time evaluated. However, they presented a fungistatic effect; the fungus continued its development after replanting in PDA medium.

For mycelial growth with respect to time, significant statistical differences were obtained (p < 0.0001), as shown in Figure 13. Lineal mycelial growth was observed in all treatments against Botrytis sp. The lowest mycelial growth occurred with PP and PP+NC treatments (Figure 8a). For Rhizopus sp., significant differences in mycelial growth were observed with respect to time. The control treatment showed mycelial growth at 10 h, and the PET treatment showed growth at 15 h. The PPCP+NC treatment showed growth at 25 h. No mycelial growth was observed in PPCP, PP, and PP+NC treatments at the evaluated times (Figure 8b).

4. Discussion

Regarding the content of volatiles present in green coffee bean oil, caffeine is a typical compound of the coffee plant characterized by having a high antioxidant activity [36]; however, caffeine is also characterized by having a broad antimicrobial activity [37]. It is present in various parts of the coffee plant. Nonthakaew et al. [38] evaluated the antimicrobial activity of caffeine extracts from coffee beans on the mycelial development of Rhizopus oryzae at concentrations of 2.5 to 10 g 100 mL⁻¹. This occurs because caffeine can inhibit fungi by stopping the synthesis of glucose, fructose, and maltose, preventing fungal spread. Other compounds such as hexadecanoic acid and octadecinoic acid with antimicrobial activity have been identified in coffee [39]. Phenolic compounds are abundant in coffee by-products, since they are characterized by their antimicrobial activity [40]. These are abundant in the CP and have the function of covering and protecting the bean from attacks by other microorganisms.

Kaempferol has been reported as a bioactive compound characterized by its high antioxidant activity and is present in various parts of the coffee plant [41]. We found kaempferol 3-O-rhamnoside present at retention time 10.7, kaempferol at retention time 15.4, and kaempferol 3-O-(6″-acetyl-galactoside) 7-O-rhamnoside at retention time 16.3, due to its wide distribution in this residue, acting as defense substances in coffee with antioxidant activity. Periferakis et al. [42], found that kaempferol was a flavonoid widely distributed in several plant species that acts as an antimicrobial agent. The antifungal activity of kaempferol can be enhanced if it is encapsulated in chitosan nanoparticles, as mentioned by IlK et al. [43], who evaluated the antifungal effect of kaempferol and kaempferol loaded lecithin/chitosan nanoparticles on Fusarium oxysporium. Using kaempferol alone resulted in 100% growth on day 20 of evaluation, while using encapsulated kaempferol resulted in a growth greater than 60% on day 60. Quercetin-3-O-galactoside and isorhamnetin inhibit bacterial division and growth during the logarithmic growth period and act as a bacterial inhibitor. Inhibition may be mediated by the disruption of the bacterial cell structure, leading to leakage of contents including sugars, nucleic acids, and proteins [44]. The antibacterial activity of theaflavin showed that epicatechin combinations against Candida albicans were stronger than that of theaflavin alone. Minimum inhibitory concentrations (MICs) of 1024 μg/mL with theaflavin and 128–256 μg/mL with theaflavin-epicatechin combinations were observed by Betts et al. [45]. The compound andrographolide has anti-inflammatory and antioxidant effects; however, it also has properties for the control of infections caused by microorganisms, due to the capacity of intracellular DNA inhibition [46,47]. In grape pomace, 2-S-Glutathionyl caftaric acid has been isolated and antimicrobial activity on three bacterial strains was reported [48]. Ficus eriobotryoides leaves were reported with antifungal activity on six isolates of Fusarium oxysporium and contained myricetin [49]. Although the CP has a wide content of compounds, these mostly act as antioxidants, which is why CP has the lowest antifungal effect compared to other treatments. Mirón-Mérida et al. [10] evaluated ethanolic extracts of CP on the mycelial growth of Colletotrichum gloesporioides and Fusarium verticillioides and found greater inhibition as the extract dose is increased; this is due to the presence of bioactive compounds, most of which are phenolic compounds. In our research the most sensitive fungus to CP was Botrytis sp. at a dose of 10% which can be attributed to the presence of the major phenolic components identified by the HPLC-MS analysis.

In the elemental analysis of CP, values of 57.72 and 56.88% of carbon were obtained, coinciding with results from Coura et al. [50], who reported a carbon content of 60.5 and 62.3% in CP because it is a lignocellulosic material rich in carbon and hydrogen. It has the capacity to release energy due to its high combustion enthalpy and this means that CP could absorb organic compounds on its surface [51].

It was reported by Cai-Ling et al. [52] that the hydroxyl group (O-H) present in the chitosan causes extracellular effects and intercellular effects, and penetrates the cell wall and the cell membrane. The presence of chlorogenic acids in the GCBO can cause early membrane permeabilization of the spores [53].

Several bioactive antifungal compounds have been identified in CP. In the in vitro tests, each treatment presented differing effects due to the differences in its bioactive constituents, but green coffee bean oil showed total inhibition in formation of spores in both fungi due to its volatile phase, which prevents its evaporation when in a closed medium such as the Petri dish. The lipophilic phase of the oil is more easily absorbed by the mycelium [54].

Chitosan has physicochemical properties that provide it with an antifungal effect. It can disintegrate the cell wall membrane of fungi, cause cytoplasm leakage, chelation of essential nutrients, and the binding of nucleic acids that alter the flow of genetic information [55]. By synthesizing chitosan as a nanoparticle, significant inhibitory effects are obtained, with changes occurring in colony formation and spore germination, and nanoparticles induce morphological changes by having a more effective membrane penetration [56]. El-Naggar et al. [57] compared the effect of chitosan and chitosan nanoparticles in inhibiting Botrytis cinerea mycelium, and showed greater inhibition when using nanoparticles. At a concentration of 1 mg/mL with chitosan, they obtained an inhibition of 31.16%, while they obtained an inhibition of 73.58% when using chitosan nanoparticles at the same concentration. This may be explained by the alteration in membrane permeability due to morphological changes that inhibit mycelial growth, sporulation, and spore germination.

In the in vivo test, blueberry fruits contain a high content of phenolic compounds and anthocyanins, which function to preserve the fruits. One way to avoid the loss of these compounds is the application of coatings, such as essential oils or nanoemulsions, that help preserve these compounds and protect the fruits against fungal attacks [58]. The CP and NC treatments presented minor incidence and severity on Botrytis sp., as did the Ch treatments on Rhizopus sp. The CP contains a high content of phenolic compounds, and NC or Ch have solution chitosan and chitosan nanoparticles, which may be responsible for this biological activity in vivo. Sun et al. [59] evaluated a coating of chitosan and carvacrol essential oil and found that the inhibitory effect against Escherichia coli and Penicillium digitatum was greater when using the treatments together than separately. Therefore, mixing a bioactive oil with chitosan can enhance its antimicrobial properties.

To evaluate the PM, the addition of active compounds in polymeric materials can serve as a controlled release vehicle against microorganisms, as reported by Black-Solis et al. [60], who evaluated the addition of cinnamon essential oil and chitosan solution to PLA and PBAT polymeric fibers. They were tested against Alternaria alternata and inhibited its mycelial growth and spore germination at doses of 6.1% cinnamon essential oil.

5. Conclusions

The in vitro effect of coffee by-products occurred mainly on the mycelial growth of Botrytis sp., with 100% inhibition in the presence of GCBO, NC, Ch, and NpCh, while in Rhizopus sp. 20% NC, Ch, and NpCh obtained 100% inhibition. For the inhibition of spore germination, GCBO was the best treatment, since it prevented spore growth at all doses. The incidence and severity in the fruits was 16 and 17% in blueberries treated with CP and NC stored at room temperature. In the in vitro evaluation, the PM showed a significant fungistatic effect on both fungi. In this work, the inhibitory effect on Botrytis sp. and Rhizopus sp. in vitro and in vivo was enhanced by incorporating coffee waste into a polymeric matrix, which could be used to produce biodegradable packaging to extend the shelf life of fruit and vegetables. The PM can be an alternative to producing active packaging since they are made with different natural components CP, NC, and GCBO, whose purpose is to release the compounds within the food packaging to avoid the presence of diseases.

The advantages of this research are that, due to the extensive production of coffee worldwide, there is a high bioavailability of these residues, which contain many compounds with antimicrobial activity. It is important for the use of different extraction methods in the future to know the potential of these residues.