Interciencia ISSN: 0378-1844 ISSN: 2244-7776 [email protected] Asociación Interciencia Venezuela

Aquino-Bolaños, Elia Nora; Cruz-Huerta, Elvia; Martín-del-Campo, Sandra T.; Herrera-Meza, Socorro; Chávez-Servia, José Luis; Acosta-Mesa, Héctor-Gabriel; Viveros Contreras, Rubí PHYSICAL AND CHEMICAL CHARACTERIZATION OF OIL THE FROM THE mexicana Marchand SEED Interciencia, vol. 44, no. 4, 2019, pp. 236-240 Asociación Interciencia Venezuela

Available in: https://www.redalyc.org/articulo.oa?id=33959119008

How to cite Complete issue Scientific Information System Redalyc More information about this article Network of Scientific Journals from Latin America and the Caribbean, Spain and Journal's webpage in redalyc.org Portugal Project academic non-profit, developed under the open access initiative PHYSICAL AND CHEMICAL CHARACTERIZATION OF OIL THE FROM THE Tapirira mexicana Marchand SEED

Elia Nora Aquino-Bolaños, Elvia Cruz-Huerta, Sandra T. Martín-del-Campo, Socorro Herrera-Meza, José Luis Chávez-Servia, Héctor Gabriel Acosta-Mesa and Rubí Viveros-Contreras

SUMMARY

In Mexican culture, seeds from endemic , such as the saponification number, peroxides and acidity index were evalu- Tapirira mexicana Marchand (locally known as duraznillo) are ated. Fatty acid profile was obtained through gas chromatogra- consumed. These seeds are eaten ripe, fresh or toasted; howev- phy-mass spectrometry. Values of humidity, rancidity, peroxides er, there is no information available about the physical charac- and acidity index were below official standard levels. Linoleic teristics and chemical composition of this seed. The purpose of acid concentration was 19 times higher than those found in M. this study was to determine the physical-chemical characteris- integrifolia. T. mexicana oil has functional characteristics ben- tics and fatty-acid profile of oil from T. Mexicana seeds. Seed eficial for human consumption, and could be used in the food dimensions and humidity were also determined. In the oil, ob- industry. To our knowledge, this is the first report on the fatty tained by pressing at 25°C, density, humidity, rancidity, color, acid composition of oil from the T. mexicana seed.

Introduction and help reduce total cholester- specifically the seeds of the physical and chemical char- ol, triglycerides and low-density Tapirira mexicana Marchand, acteristics and the fatty acid Fatty acids are fundamental lipoproteins, while increasing known locally as duraznillo, a profile of oil from Tapirira nutrients in the human diet giv- high-density lipoproteins native species to Mexico that is mexicana Marchand seeds. en the vital functions they per- (Vadivel et al., 2012). The cur- eaten in the states of , form in the body: they consti- rent focus of food-related stud- Oaxaca, Puebla and Veracruz Materials and Methods tute the main energy reserve, ies tends to be on products with (Lascurain et al., 2010). transport a range of nutrients, high levels of MUFA and Seeds are picked by members Species regulate metabolism (Badui, PUFA. As such, the USA Food of the local communities and 2012), are a structural part of and Drug Administration (FDA) sold in local markets, consumed Tapirira mexicana March- cell membranes (Levant et al., has indicated that eating nuts is ripe, fresh or toasted (using lo- and is a genus of neotropical 2004), and regulate several an important source of MUFA cal, artisanal techniques) from small plants belonging to brain functions (Lee, 2013), and essential fatty acids, and July to September (Penington the family; it is among others. that the regular consumption of and Sarukhán, 2005). However, found in Mexico and parts of As such, it is important to nuts has beneficial health ef- despite being a common prac- South America, mainly in incorporate monounsaturated fects (Alasalvar and Shahidi, tice in the region, to our knowl- Peru, Bolivia, Paraguay and fatty acids (MUFA) and poly- 2009). edge no studies have been re- Brazil (Wendt and Mitchell, unsaturated fatty acids (PUFA) Furthermore, it is a common ported regarding the physical 1995; Terrazas and Wendt, into the diet, given that they cultural practice among commu- and chemical characteristics of 1995a, b). The genus encom- can decrease the risk of devel- nities in Mexico to eat seeds the duraznillo seeds. The goal passes both trees and shrubs, oping cardiovascular diseases from endemic plants, of this study was to evaluate with the reaching a height

KEYWORDS / Fatty-Acid Profile / Linoleic Acid / Physical-Chemical Analysis / Seed Oil / Received: 09/20/2018. Modified: 05/02/2019. Accepted: 05/03/2019.

Elia Nora Aquino-Bolaños Elvia Cruz-Huerta. Ph.D. in Science, UV, Mexico. Doctor in ITVer, Mexico. M.Sc. in (Corresponding autor). Chemical Biochemistry, Molecular Biology Food Science, Instituto Artificial Intelligence, UV, Engineer, Instituto Tecnológico and Biomedicine, Universidad Tecnológico de Veracruz Mexico. Doctor in de Oaxaca, Mexico. Doctor in Complutense de Madrid. Spain. (ITVer), Mexico. Professor, UV, Artificial Intelligence, Food Science, Universidad Research Professor, UV, Mexico. University of Sheffield, UK. Autónoma de Querétaro, Mexico. José Luis Chávez-Servia. Doctor Research Professor, UV, Mexico. Research Professor, Sandra T. Martín-del-Campo. in Genetics, Colegio de Mexico. Universidad Veracruzana (UV), Doctor in Science, Universidad Posgraduados, Mexico. Rubí Viveros Contreras. Mexico. Address: Instituto de de Guadalajara, Mexico / Professor-Researcher, Centro In- Nutritionist, UV, Mexico. Ciencias Básicas, Universidad Institut National Agronomique terdisciplinario de Investigación Master in Food Science, Veracruzana. Av. Dr Rafael Paris-Grignon, France. Research para el Desarrollo Integral UV, Mexico, Doctor in Sánchez Altamirano s/n Col Professor, Tecnológico de Regional Oaxaca, Instituto Food Science, IPN, Mexico. Industrial Animas, 91192, Monterrey, Mexico. Politécnico Nacional, Mexico. Research Professor, UV- Xalapa, Veracruz. Mexico. Socorro Herrera-Meza. Nutri- Héctor-Gabriel Acosta-Mesa. CONACyT, Mexico. e-mail: [email protected] tionist and M.Sc. in Food Computer Systems Engineer,

236 0378-1844/14/07/468-08 $ 3.00/0 APRIL 2019 • VOL. 44 Nº 4 CARACTERIZACIÓN FÍSICA Y QUÍMICA DEL ACEITE DE LA SEMILLA DE Tapirira mexicana Marchand Elia Nora Aquino-Bolaños, Elvia Cruz-Huerta, Sandra T. Martín-del-Campo, Socorro Herrera-Meza, José Luis Chávez-Servia, Héctor Gabriel Acosta-Mesa y Rubí Viveros-Contreras RESUMEN En México se consumen semillas de plantas endémicas grasos se obtuvo mediante cromatografía de gases acoplado como parte de la cultura. Ejemplo de esto es la semilla del a espectrometría de masas. El porcentaje de humedad, densi- duraznillo (Tapirira mexicana Marchand) cuyas semillas son dad, peróxidos e índice de acidez se encontraron por debajo consumidas maduras, frescas o tostadas. Sin embargo, no se de lo establecido en las normas oficiales. Se identificaron los tiene información disponible sobre las características físicas ácidos grasos en el aceite, encontrando al ácido linoleico 19 y composición química de esta semilla. El propósito de este veces más elevado en comparación con el aceite de M. inte- estudio fue determinar las características físico-químicas y el grifolia. El aceite de la especie de T. Mexicana tiene caracte- perfil de ácidos grasos del aceite de semillas de T. mexica- rísticas funcionales benéficas para el consumo humano por lo na. Las dimensiones y humedad de las semillas también fueron que podría utilizarse en la industria alimentaria. Hasta don- determinadas. En el aceite, obtenido por prensado a 25 °C, de conocemos, este es el primer reporte sobre la composición se evaluó densidad, humedad, rancidez, color, índice de sa- de los ácidos grasos del aceite de la semilla de la especie T. ponificación, peróxidos e índice de acidez. El perfil de ácidos Mexicana.

CARACTERIZAÇÃO FÍSICA E QUÍMICA DO ÓLEO DE SEMENTE DE Tapirira mexicana Marchand Elia Nora Aquino-Bolaños, Elvia Cruz-Huerta, Sandra T. Martín-del-Campo, Socorro Herrera-Meza, José Luis Chávez-Servia, Héctor Gabriel Acosta-Mesa e Rubí Viveros-Contreras

RESUMO

No México se consomem sementes de plantas endêmicas de ácidos graxos foi obtido mediante cromatografia de gases, como parte da cultura. Exemplo disto é a semente da Tapirirá acoplado a espectrometria de massas. A porcentagem de umi- (Tapirira mexicana Marchand) cujas sementes são consumidas dade, densidade, peróxidos e índice de acidez foram observa- maduras, frescas ou torradas. No entanto, não há informação dos abaixo do estabelecido nas normas oficiais. Identificaram- disponível sobre as características físicas e composição quími- -se ácidos graxos no óleo, o ácido linoleico foi encontrado 19 ca de esta semente. O propósito de este estudo foi determinar vezes mais elevado em comparação com o óleo de M. integri- as características físico-químicas e o perfil de ácidos graxos folia. O óleo da espécie de T. Mexicana tem características do óleo de semente de T. mexicana. As dimensões e umidade funcionais benéficas para o consumo humano por isto poderia das sementes também foram determinadas. No óleo, obtido por utilizar-se na indústria alimentar. Até onde conhecemos, este é prensagem a 25 °C, se avaliou densidade, umidade, ranço, cor, o primeiro relatório sobre a composição dos ácidos graxos do índice de saponificação, peróxidos e índice de acidez. O perfil óleo da semente da espécie T. Mexicana.

of 30m, producing a wood Sample preparation calculated according to AOAC total lipids content, rancidity, similar to mahogany that is (1999). acidity index, saponification used in furniture. Its flowers The fruits of T. mexicana number, density, humidity, co- are small, inflorescent and pa- were collected completely ripe Physical and chemical lor parameters and identifica- niculate, and the fruit is fleshy and free of mechanical dam- parameters of the oil tion and quantification of oil and ovoid in shape, reaching age, and the pulp and shell fatty acids. 2cm in length (Penington and were removed. The seeds were Oil extraction. The extraction The total lipids content was Sarukhán, 2005). dried for 60 days at room tem- of the oil was carried out by measured using the Soxhlet perature before removing the grinding 500g of shelled du- 930.39 method from AOAC Sample collection pericarp, prior to further raznillo seeds to obtain a pas- (2000) using petroleum ether analysis. te, which was then compressed for extraction. Rancidity, acid- The fruits of T. mexicana under a weight of 10t for 1min ity index, saponification num- were collected randomly Physical characteristics of the using a hydraulic press ber, density and humidity test from Rancho Plan de San seed (PowerTeam, mod. A, USA). were determined using the cor- Antonio, located on the The oil obtained was centrifu- responding Mexican standard high ground surrounding the To measure the length and ged at 8000 rpm for 20min guidelines (NMX-F-222- town of Coatepec, Veracruz, width of the seed and the (Biofuge Primo R, Heraeus, SCFI-1995, NMX-F-075- Mexico (19°28'47.05''N, thickness of the pericarp, a USA) to eliminate any solid SCFI-2006, NMX-F-174- 96°59'28.1''W, altitude of ±0.01mm digital vernier residue. The supernatant was SCFI-2006, NMX-F-211- 1,524masl). Average annual (Mitutoyo, mod. Absolute placed in amber colored jars SCFI-2006, NMX-F-101- precipitation for the area is Digimatic, Japan) was used. using nitrogen gas to eliminate SCFI-2012), while the peroxide 1,035mm, and average annu- The percentage of seed without any air. The oil was stored at number was determined in al temperature is 16-22°C. shell and the humidity were -20°C for further analysis of compliance with AOAC (2000).

APRIL 2019 • VOL. 44 Nº 4 237 The oil color was evaluated the corresponding methyl es- ±0.13%) compared to the oil, the threshold of which is using a spectrophotometer ters of fatty acids (Sigma- Macadamia integrifolia nut, 15.0mEq O2 active/kg oil (Konica Minolta, mod. CM- Aldrich). Quantification was which has reported values of (CODEX, 2009). The average 2500d). The parameters L*, a* achieved by using the relative 3.48 ±0.27% (Mapel, 2014). value found is within the range and b* were used to calculate area percentage method. All When comparing the percent- reported by Kaijser et al. chromaticity (C*= (a*2+b*2)1⁄2) samples were analyzed in age of seed minus the shell it (2000) for the oil of different and hue angle (h°= tan-1(b*⁄a*)) quadruplicate. was observed that this level is species of Macadamia tetrap- (McGuire, 1992). similar to that reported for M. hylla (0.56-3.61mEq O2/kg), but Statistical analysis integrifolia (30.6 ±0.28% vs is higher than that reported for Identification and quantifica- 30.31%, respectively) (Mapel, different species of almond tion of fatty acids. Fatty acids The comparison of the statis- 2014). (0.34-0.43mEq O2/kg oil; were derived based on tical significance of the aver- Moayedi et al., 2011) and pea- Hernández-Galán (2011), using age values obtained in the du- Physical and chemical nut (1.01 mEq O2/kg oil; chloroform and sodium raznillo seed oil with the parameters of the oil Özcan, 2010). methoxide. Fatty acid methyl benchmark values of the maca- The acidity index of an oil esters (FAME) were analyzed damia nut commonly con- Total lipids content of T. indicates its free fatty acid using a gas chromatograph sumed in the same region was mexicana was 73.10g/100g dw content, so the lower the free (Agilent mod. 7890A) and cou- done using the t-Student test. (dry weight), a similar value to fatty acid content, the better pled to a mass detector The statistical analysis was that in walnut (75.4g/100g; Li the oil quality is (Badui, 2012; (Agilent mod. 5975 C). The carried out using Minitab 17 and Hu, 2011) and within the Ibeto et al., 2012). T. mexicana capillary column used was an software. range reported by Aquino- oil has an acidity index of HP-88 (Agilent) Bolaños (2017) for different 1.4mg KOH/g oil (Table II), 100m×0.250mm×0.20µm. The Results and Discussion species and hybrids of macada- this value is in keeping with conditions were described by mia (70.9 to 79.7g/100g). international regulations for Aquino-Bolaños et al. (2017). However, the value is higher crude cold-pressed oil, the Helium was used as a carrier Physical parameters of the than that of the fat content of threshold of which is 4mg gas and mass spectra were ob- seed nuts of common consumption, KOH/g (CODEX, 2009). The tained through electron-impact such as almonds value for T. mexicana oil is ionization at 70eV. The characterization of (44.4-51.4g/100g; Moayedi et lower than that reported for oil Identification of the peaks for Tapirira mexicana Marchand al., 2011), pistachios (53.9g/100; from different varieties of pea- each fatty acid was carried out seeds is shown in Table I. The Li and Hu 2011) and peanuts nut (3.96-4.95mg KOH/g oil; using MSD ChemStation seeds were found to be smaller (32.7-45.4%; Özcan, 2010). Shad et al., 2012), but higher E.02.00.493 software (Agilent) than those reported for this The physical and chemical than that reported for the oil of and the National Institute of species by Sugiyama and properties of T. mexicana seeds different species of almond Standards and Technology Peterson (2013) (13.3 ±0.13mm are shown in Table II. The per- (0.26-0.30mg KOH/g oil; (NIST) database, and the iden- and 9.5 ±0.18mm vs 17.3 ±0.17 centage of humidity and vola- Moayedi et al., 2011) and M. tity of the most important fatty and 11.3 ±0.12mm, respective- tile materials for T. mexicana integrifolia (0.820mg KOH/g acids confirmed by the reten- ly). As to humidity, T. mexi- oil was found to be of 0.17 oil; Rodríguez et al., 2011). tion time and mass spectra of cana showed higher values (6.3 ±0.02%, indicating that it has a The saponification number is low water content, which fa- expressed as the number of mg TABLE I vors the stability (from oxida- of KOH required to saponify CHARACTERIZATION OF Tapirira mexicana Marchand tion) of the oil during storage. 1g of the sample (Nayak and SPECIES SEED This value is higher than that Patel, 2010). This value is in- Characteristics reported for the oil from M. versely related to the average Humidity (%) 6.3 ±0.13 integrifolia nut, of 0.085% molecular weight of the fatty (Rodríguez et al., 2011). acids (Badui, 2012). In this Seed without shell (%) 30.6 ±0.28 The density of fatty acids study, the saponification num- Pericarp thickness (mm) 2.3 ±0.07 and glycerides increases as its ber for T. mexicana oil was Length (mm) 13.3 ±0.13 molecular weight decreases and 165.2 ±0.86mg KOH/g oil, a Width (mm) 9.5 ±0.18 its degree of unsaturation in- similar value as that reported Values expressed as averages ±SD. creases (Badui, 2012). The for peanuts (165.3-187.6mg density of T. mexicana oil was KOH/g oil; Özcan, 2010). This TABLE II found to be 0.90g·ml-1, lower number is higher than that for PHYSICAL AND CHEMICAL PARAMETERS OF Tapirira than reported for the oil of cashew kernel oil (137mg mexicana Marchand OIL different varieties of peanuts KOH/g oil; Akinhanmi et al., (0.915-0.918g·ml-1; Shad et al., 2008) and lower than that for Parameters 2012) and M. integrifolia M. integrifolia (233.656mg Total lipids content (%) 73.1 ±0.17 (0.9116g·ml-1; Rodríguez et al., KOH/g oil; Rodríguez et al., Humidity (%) 0.17 ±0.02 2011). 2011). The low saponification Density (g/ml) 0.90 ±0.00 Peroxides are the main prod- number reported for T. mexi- Peroxide value (mEq O2/kg) 2.2 ±0.05 ucts of primary autoxidative cana is a sign that the oil is Acidity index (mg KOH/g) 1.4 ±0.00 degradation. T. mexicana oil not appropriate for soap manu- showed an average value (2.2 facturing. The rancidity test for Saponification number (mg/g) 165.2 ±0.86 ±0.05mEq O2/kg oil) below the T. mexicana was negative. Rancidity Negative standards of international regu- Evaluating the color of oil is Values expressed as averages ±SD. lations for crude, cold-pressed necessary given that the

238 APRIL 2019 • VOL. 44 Nº 4 appearance of the product is mexicana seed (41% and 74%, TABLE IV the main element that a con- respectively; Table IV). FATTY ACID PROFILE FROM Tapirira mexicana VS sumer evaluates prior to pur- The seed of T. mexicana Macadamia integrifolia OIL chasing it. In this study, T. contains a high percentage of Fatty Acids T. mexicana M. intergrifolia p-value mexicana oil is shown to have monounsaturated fatty acids a clear, bright yellow tone with and polyunsaturated fatty acids Palmitic, C16:0 18.5 ±0.18 13.1 ±1 0.00 values of 11.4 ±1.32 for param- (Table IV). The specific spe- Palmitoleic, C16:1 1.9 ±0.55 24.0 ±2 0.00 eter b* and 2.7 ±0.32 for a*. cies studied in this research The values for C* and hº, in- project contains a higher pro- Stearic, C18:0 10.6 ±1.67 6.1 ±0.8 0.00 dicatives of chroma and hue, portion of oleic fatty acids Oleic, C18:1 37.2 ±0.25 45.0 ±4.0 0.03 were 11.7 ±1.26 and 76.7 ±2.48, (C18:1), the consumption of Linoleic, C18:2 31.0 ±0.62 1.6 ±1.6 0.00 respectively (Table III). In gen- which has been associated with α-Linolenic, C18:3 0.1 ±0.01 0.2 ±0.02 0.99 eral, it is preferable that edible a decreased risk of cardiovas- oils be translucent and clear in cular disease, arrhythmia and Arachidic, C20:0 0.6 ±0.02 3.9 ±0.4 0.00 color. The oil under study has stroke, among others (Morón, Eicosenoic, C20:1 0.1 ±0.02 3.5 ±0.4 0.00 characteristics that are desir- 2008; Gil and Serra, 2013). Behenic, C22:0 0.1 ±0.01 1.3 ±0.2 0.00 able for consumers. Regarding linoleic acid (C18:2), levels in T. mexicana are 19 Lignoceric, C24:0 0.1 ±0.03 0.4 ±0.04 0.99 Fatty acid composition times higher when compared to SFA 29.9 ±1.68 24.8 ±1.33 0.00 M. integrifolia. This essential MUFA 39.1 ±0.60 72.5 ±4.39 0.00 Ten fatty acids were identi- fatty acid helps metabolyze fied in T. mexicana seed oil, of polyunsaturated fatty acids, is PUFA 31.0 ±0.63 1.8 ±1.6 0.00 which oleic, linoleic, palmitic a necessary element of cutane- MUFA/SFA 1.3 ±0.07 2.9 ±0.24 0.00 and stearic acids were found in ous fat and its consumption is Values are expressed as averages ±SD. The averages for Macadamia the greatest proportions, corre- associated with decreased cor- integrifolia were reported by Aquino-Bolaños et al. (2017). The statistical sponding to 97.3% of the total onary risk (Gil and Serra, significance of the differences between the averages was evaluated using fatty acids (Table IV). 2013; Mataix and Gil, 2004). statistical evidence of t-student. Individual fatty acids were calculated as Several studies have reported It has been reported that the the percentage of total fatty acids. SFA: saturated fatty acids, MUFA: that saturated fatty acids, such consumption of palmitoleic monounsaturated fatty acids, PUFA: polyunsaturated fatty acids as stearic acid (C18:0), have a acid (C16:1) is associated with positive impact on human cardiac arrhythmia (Oyanagi et health, given that they have al., 2015) and mortality due to Conclusions and cashew nut shell liquid. J. antioxidant, anti-inflammatory cardiovascular issues Agric. Food Environ. Sci. 2: (Nanji et al., 2001; Wang et (Ebbesson et al., 2010). In this Oleic, linoleic, palmitic and 1-10. al., 2007; Pin-Ho et al., 2010), study, the amount of palmitole- stearic acids were identified in Alasalvar C, Shahidi F (2009) Tree hepatoprotective, decreasing ic acid (C16:1) was 92% lower the Tapirira mexicana Nuts: Composition, hepatic lesions caused by cho- than to that reported for M. Phytochemicals, and Health Marchand seed. The fatty acid Effects. CRC. New York, USA. lesterol (Pin-Ho et al., 2010) integrifolia. In terms of C18:3 profile indicates that T. mexi- 340 pp. and neuroprotective (Wang et (α-linoleic) content, there is no cana seeds have functional Aquino-Bolaños EN, Mapel-Velazco al., 2007) effects, in addition significant difference between benefits for human consump- L, Martín-del-Campo ST, to decreasing blood cholesterol T. mexicana and M. integrifolia tion. Furthermore, the ex- Chávez-Servia JL, Martínez AJ, levels (Ruddle et al., 2013). As (Table IV). traction of the oil from this Verdalet-Guzmán I (2017) Fatty such, when comparing the fatty The MUFA/SFA index is seed could be useful for indus- acids profile of oil from nine acid profiles of T. mexicana used to evaluate the nutritional trial purposes, given the physi- varieties of macadamia nut. Int. J. Food Prop. 20: 1262-1269. and M. integrifolia (Aquino- value and the healthy con- cal and chemical characteristics Bolaños et al., 2017) nuts, it sumption of fat (Mamani- of the oil. According to our AOAC (1999) Official Methods of can be seen that palmitic Linares and Gallo, 2013). Analysis. Association of Official knowledge, this is the first re- Analytical Chemists. http:// (C16:0) and stearic (C18:0) fat- According to the results ob- port on the fatty acid composi- kb.psu.ac.th:8080/psukb/bits- ty acids were found in greater tained in this study, the tion of the T. mexicana tream/2553/1573/2/271250_app. concentrations in the T. amount of monounsaturated Marchand seeds. pdf (Cons. 18/06/2017). fatty acids (MUFA) and satu- AOAC (2000) Official Methods of rated fatty acids (SFA) found ACKNOWLEDGEMENTS Analysis. Association of Official in the seed of T. mexicana are Analytical Chemists http://we- TABLE III significantly different when Biological material was pro- bpages.icav.up.pt/PTDC/CVT- compared to those reported for NUT/4294/2012/AOAC%202000. COLOR PARAMETERS OF vided by Unión de Productores pdf (Cons. 18/06/2017). Tapirira mexicana Marchand M. integrifolia (Table IV). The Agropecuarios Forestales de la amount of polyunsaturated fat- Badui DS (2012) Química de los OIL Zona Alta de Coatepec, Alimentos. Person. México. 648 ty acids (PUFA) is 17 times Veracruz, A.C., Mexico. pp. Color parameter higher in T. mexicana seeds L* 29.8 ±1.02 when compared to M. integri- CODEX (2009) CODEX STAN 210- REFERENCES 1999. 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