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Production of Functional Ice Cream Fortified by Immunoglobulin Y against Escherichia coli O157:H7 and Helicobacter Pylori

: The production of effective functional foods that can increase public health is paramount to scientists. This study aimed to optimize methods for developing bio-ice cream comprising IgY versus Escherichia coli O157 and Helicobacter pylori . Leghorn hens were immunized intramuscularly with antigens. The egg yolk was separated from the egg, and the IgY antibody was then purified using the PEG 6000 method. Maximum IgY was reached in 2 weeks with IgY concentrations of 14.56, 11.46, 16.34, and 6.87 mg/ml for H. pylori , E. coli O157, E. coli + H. pylori , and control, respectively. The results showed that 0.6 mg/ml concentration of IgY had a significant inhibitory effect on bacteria growth using disk diffusion methods. Also, ELISA results showed that IgY activity was reliable and remained active in ice cream for 3 months. According to sensory evaluation, the global acceptance scores and the other attributes revealed the acceptable acceptance of IgY egg yolk ice cream. Consequently, it can be stated that the ice cream matrix can be considered an ideal candidate for carrying antibodies into the host body, as the specific ingredients of the ice cream matrix can appropriately protect IgY antibodies.

Introduction
In recent years, biologically active components have been regarded as one of the most crucial factors in food quality [1][2][3]. Scientific research has suggested that functional foods could be effectively used for disease prevention in humans [4][5][6]. However, the application of antibiotics in food-generating animals as simple candidates for this purpose has given serious cause for concern considering their potential risks to human health [7,8]. Recently, IgY or egg yolk-related antibodies have attracted scientific consideration as potential alternatives for antibiotics to encourage growth in the attendance of disease-triggering agents [9,10]. Compared to mammalian IgG, IgY has numerous benefits, including cost-effectiveness, high yield, and convenience [11]. It has been previously demonstrated that the oral consumption of IgY antibodies could provide considerable protection against various intestinal pathogens (e.g., Helicobacter pylori, enterotoxigenic Escherichia coli, Mycobacterium tuberculosis, Clostridium difficile, Salmonella typhimurium, Acinetobacter baumannii, Prevotella intermedia, Fusobacterium nucleatum, Propionibacterium acnes, and Vibrio spp) [12].
E. coli O157:H7 and H. pylori are among the top harmful microorganisms that threaten human health [13,14]. In addition to being the well-known reason for gastritis and gastric ulcers, H. pylori possess a significant role in the expansion of gastric carcinomas, and about 50% of the global human population has been affected by this bacterium [15,16]. Moreover, about 10% of children with an E. coli O157:H7 infection will suffer from hemolytic uremic syndrome (HUS), with an annual 5% mortality rate. The children who survive will possess a higher risk for emerging chronic kidney disorder [17]. Also, it has been stated that the application of IgY versus E. coli O157:H7 and H. pylori can be predominantly valuable for diseases prevention and treatment strategies [18][19][20]. The growth inhibitory function of the IgY antibody is one of its vital characteristics, meaning that the IgY antibody could be intake in a daily diet with no significant side effects. Also, it could contribute to regulating the intestinal microbial population of the host [21][22][23]. Several studies have previously focused on adding IgY to yogurt; however, their results have not been commercialized, partly since, in their study, the type of food matrix (yogurt) reduces the concentration of antibodies [24]. Moreover, NaCl and other components of yogurt had negative effects on the stability of IgY proteins.
Hence, in the present study, the feasibility of the production of egg-ice cream containing IgY antibodies against E .coli O157:H7 and H. pylori was examined for the first time. If the results are positive and effective, this formulation can be used to develop functional ice cream and improve the health status of people in the community.

Chickens.
Sixteen white leghorn chickens 13 weeks old were purchased from a local hatcher (Animal science department of the Ferdowsi University of Mashhad). The chickens were kept under environmental conditions of 18 h light and a free diet [25].

Preparation of antigen.
E. coli O157:H7 and H. pylori were obtained from the Department of Microbiology of Ghaem Hospital (Mashhad, Iran). Sorbitol-MacConkey and Mueller-Hinton broth media were used for E. coli O157:H7 and H. pylori suspension. Then, 10 9 CFU/ml of each bacterium were washed with 5ml NaCl 85% (3 times) and centrifuged for 10 min at 10,000 rpm. For bacteria inactivation, pellets were suspended in 0.3% formaldehyde and then centrifuged for 10 min at 10,000 rpm. The pellets were re-suspended in 5ml NaCl 85% and stored at 4°C.

Immunization of hens.
Four groups of chickens (n=3 in each group) were immunized, and as the first immunization, they received an injection of E. coli O157:H7, H. pylori, and a combination of E. coli O157:H7, H. pylori with an equal volume of Freund's complete adjuvant (Sigma, USA) when they reached 16 weeks of age. Also, Freund's complete adjuvant without antigen was injected into the control group, followed by two booster immunizations utilizing incomplete Freund's adjuvant at a two-week interval. The last injection of chickens was done, and about a week later, the eggs were collected daily for eight weeks, and after labeling, they were kept at 4 °C [26].

Extraction and purification of IgY.
Extraction and purification of IgY antibody from egg yolk were done using polyethylene glycol 6000 (PEG 6000) powder (Merck, Germany) according to Polson's method [26]. After separating the egg yolk, phosphate-buffered saline (twice the volume of yolk suspension, pH=7.6) was added and before mixed, followed by the addition of the PEG 3.5% (w/v). The resulting solution was shaken at room temperature for 10 minutes and finally centrifuged (13000 g, 20 min, 4 °C). To remove lipids, the supernatant was collected and filtered using Whatman filter paper. PEG 6000 was added to 8% (w/v) concentration. The shaking was then performed at room temperature for 10 minutes and centrifuged again as previously described. To dissolve the precipitation, 10 ml PBS and 12% (w/v) PEG 6000 were added and centrifuged as earlier. The pellet with IgY was re-suspended in 2 ml PBS and purified by dialysis against PBS overnight (4 °C). The IgY was investigated by the SDS-PAGE and Bradford methods.

ELISA assay for activity detection.
The specific activity of the IgY antibody against E. coli and H. pylori was examined by ELISA. 100 μL of the antigens was adsorbed on microtiter plates (10 3 , 10 4 , 10 5 , and 10 6 CFU/ml) for 1 h. The plates were then washed three times using PBS with 0.05% (v/v) Tween 20 (PBST). Using blocking buffer (1.5% BSA in PBST), nonspecific sites were blocked for 1 h at 37 °C. After being washed, we added the primary antibody, which consisted of 100 μL of diluted IgY antibody (1:2000) in PBST. Subsequently, it was incubated for 1 h at 37 °C. Afterward, the plates were re-washed, and 100 μL of rabbit anti-chicken IgY H&L (HRP) secondary antibody (Sigma, USA) at a diluted IgY antibody (1:10000) was added to each well and before incubated at 37 °C for 1 h. To perform colorimetric detection, O-phenylenediamine (OPD, Sigma) was used as a chromogenic substrate of HRP. Finally, wells were washed using PBST. In order to stop the reaction, 50 μL/well 2N H2SO4 was added. The absorbance of each well was measured at 492 nm [25].

Estimation of IgY cytotoxicity by disk diffusion method.
One of the simple tests for cytotoxicity of antibodies has been the investigation of bacteria growth in the presence of antibodies. The 5 different concentrations of IgY were prepared and injected into disks. 10 µl of E. coli and H. pylori suspension were cultured on specific media, and disks were placed on it. Gentamycin (10µg/ml) antibiotic was used as control.

Preparation of egg ice cream.
The ice cream preparation was done according to Herald et al. [27] Briefly, egg yolks containing IgY were used in the ice cream formula. 1.46 g egg yolks, 0.20 g stabilizer, 1.36 g nonfat dry milk, 6.05 g sugar, 23.58 g pasteurized milk, and 9.07 g cream were polled and stirred for 10 min at 70 °C. The suspension was cooled to 32°C and homogenized at 1500 psi. The homogenized suspension was cooled to 4°C immediately and tempered for 24 h at 4°C. Finally, ice cream was produced by freezing ice cream at -18 °C. The residual activity of IgY after freezing was evaluated by ELISA for 3 months. Ice cream containing IgY (5 mL) was combined with a similar amount of carbonate buffer (pH=10) and mixed for 3 min with chloroform (10 mL). The solution was centrifuged at 3000 rpm for 30 min, after which we examined the upper supernatant for IgY shelf life in ice cream using ELISA, as discussed earlier [24].

Sensory evaluation of the ice creams.
Sensory assessment of the egg yolk ice creams was conducted through surveys to evaluate consumers' level of acceptance. Within two weeks of storage, the surveys were carried out on the samples, through which participants were questioned to grade the ice creams based on color, texture, flavor, feel, taste, and global acceptance, using a 5-point hedonic scale [28].

Statistical analyses.
The results were expressed as the means ± standard deviation (SD) for data in each shown treated group. Student t-tests were applied to recognize statistically significant differences between the two groups. Differences were considered significant at p < 0.05.

Production and purification of IgY.
The purification of IgY using the SDS PAGE methods was investigated and displayed in Figure 1. Results showed that IgY purification from 4 samples (E. coli, H. pylori, E. coli + H. pylori, and control) was done correctly. The size of heavy and light chains of IgY was estimated at 67 and 35 kDa, respectively. Also, our results confirmed that IgY purity was satisfactory.
Furthermore, IgY yield in samples was 14.56, 11.46, 16.34, and 6.87 mg/ml for E. coli, H. pylori, E. coli + H. pylori, and control, respectively. Therefore, considering the optimal purity and high efficiency of IgY, it can be utilized as a novel biological strategy and an effective and ideal type of antibody in supplying the food safety chain in the food industry. Among the most important factors in functional food production are the yield and purification of IgY, which requires optimization for each experiment [21]. The purification of IgY from egg yolk by PEG methods is remarkably cost-effective and consists of two stages [26]. In stage 1, lipids were removed, and in stage 2, IgY was precipitated in PBS buffer. Our results showed that stage 1 had a major role in the final concentration of IgY. Amro et al. [21] extracted IgY using a lowpressure chromatography device and reported 3.66 mg/ml IgY for Single Comb White Leghorns egg yolk, whereas our extraction of IgY was about 2.59 mg/ml, which is suitable.

IgY activity.
ELISA assay showed binding of IgY to H. pylori and E. coli antigen that was coated on plates ( Figure 2a). Besides, the outcomes discovered significant differences between control and treatment groups (P-value <0.001). The concentration of IgY in the E. coli + H. pylori group was greater than that of the treatment group. Complete immunization was obtained two weeks after antigen injection (Figure 2b). Our results showed that the highest level of IgY purification is achieved 2 weeks after the injection. However, the decrease in IgY production was insignificant over the following weeks. Studies showed that using an appropriate adjunct and accurate estimation of antigen concentration is crucial to the production of desired results [25,29].

Cytotoxicity of IgY.
The effect of the IgY antibody on the growth of E. coli and H. pylori was measured, and the results confirmed that, similar to gentamicin, IgY could be effective at 6 mg/ml concentration. The formation of a clear zone on the disk indicated the inhibitory effect of IgY on bacteria growth (Figure 3). In light of the limited number of studies on the use of egg yolk in ice cream, our results are rather new and suggest that 0.6 mg/ml of IgY is needed for desirable effectiveness against investigated bacteria. On the other hand, pepsin and other molecules in the human stomach had an inhibiting effect on IgY activity [12] . In a study by Pauly et al. [26], 80% purity in the extraction of total IgY from egg yolk was achieved using PEG precipitation. Therefore, we condensed IgY by freeze-drying the egg yolks and then used it in ice cream preparation. The concentration of IgY was ~ 1.5 mg in 100 g of ice cream. ELISA analysis showed that the freeze-drying process had no significant effect on IgY activity. IgY shelf life appeared to be acceptable after 3 months, and IgY activity was not affected by time and mechanical activity.

Properties of ice cream.
The shelf life results of IgY in the matrix of ice cream exhibited that this dairy-based matrix could be most certainly applied as a novel functional food.  ELISA analysis established that IgY could remain active in the ice cream matrix for 3 months (Figure 4). The activity of IgY diminished by about 6%, which is insignificant. Nevertheless, studies have demonstrated that even broken IgY can act effectively against bacteria [10]. As we used a 1.5 mg/ml concentration of IgY in ice cream, the taste and other properties of the ice cream were not influenced.

Sensory analysis of ice cream.
A hedonic scale's sensory characteristics of ice cream demonstrated that this commodity could be an appropriate option for marketing, as no substantial alterations were detected between egg yolk and vanilla ice creams (Table 1). Comparing the global acceptance scores and the other attributes revealed the acceptable acceptance of IgY egg yolk ice cream. IgY containing ice cream is deemed the foremost candidate for the formulation and production of novel functional foods. It possesses the characteristics required to preserve IgY and raise the effectiveness of IgY activity. An ideal temperature for the maintenance of ice cream was -18 °C which is utterly appropriate for IgY protection. Besides, ice cream is consumed while cold, and various studies displayed that IgY can continue effective at temperatures of up to 80 °C, and the pasteurization process did not influence IgY function [24]. However, we used pasteurized milk to tackle this problem in this study. Jaradat et al. [30], reported that, in the presence of carbohydrates (sucrose, lactose, and trehalose), IgY had insignificant activity. Hence, we utilized freeze-dried egg yolk to preserve IgY biological function. Consequently, it can be stated that the ice cream matrix can be considered an ideal candidate for carrying antibodies into the host body, as the specific ingredients of the ice cream matrix can appropriately protect IgY antibodies.

Conclusions
Our results demonstrated that pathogenic microbial antigens could stimulate a humoral immune response in Leghorn hens, resulting in a synthesis of high specific immunoglobulins against E. coli O157:H7 and H. pylori, which these specific immunoglobulins according to the ELISA assay possess a significant activity in the investigated egg ice cream matrix during 3 months. IgY can be used as a significant substitute to mammalian antibodies. It is better to immunize hens before they initiate egg-producing since the stress prompted via handling them could have a side effect on egg production, as might the nature of the antigen or adjuvant have applied. Furthermore, according to the outcomes of sensorial analysis and IgY activity in the egg ice cream, it can be concluded that the ice cream can be an appropriate candidate for transferring antibodies to the human body. It is expected that the utility of IgY technology and its widespread utilization in research and medicine will increase on a large scale. IgY is expected to play an increasingly important role in future research, diagnosis, and immunotherapy.


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

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Tapioca quality observation based on physical and chemical properties of products

PT Sinar Pematang Mulia, located in the Mataram Udik area, Central Lampung, is a company engaged in the tapioca flour industry with a production capacity of 500 tons/day. The purpose of the observation in this practical work is to determine whether the tapioca flour product of PT Sinar Pematang Mulia II is following the SNI for the quality of tapioca flour. The methods used include data collection and interviews. The analysis carried out as a determinant of the quality of the finished goods product is the analysis of water content, pH, retain a sample of 100 mesh, impurities mesh 325, SO 2 , ash content, starch content, whiteness, acid degree, and viscosity. Data were collected by analyzing for seven days and comparing the value of the analysis results with the standard of SNI tapioca flour and PerKBPOM no. 36 of 2013. It can be seen that in all the results of the analysis carried out, none of them exceeds the maximum limit.

INTRODUCTION
In Indonesia, cassava is the second largest agricultural food production after rice, so cassava has the potential as an important raw material for various food and industrial products. As human food, cassava has several deficiencies, including low protein and vitamins and unbalanced nutritional value. Cassava or cassava is one of the food sources of carbohydrates (a source of energy). Cassava in a new state does not last long. For marketing that takes a long time, cassava must be processed first into other forms that are more durable, such as cassava, tapioca (cassava flour), tapai, peuyeum, cassava chips, and others (Koswara, 2013). Tapioca flour is starch extracted with water from cassava tubers. Tapioca is widely used as a thickening agent, filler, and binder in the food industry (Astawan, 2009).
Supervision and quality control are essential factors for a company to maintain consistency of product quality, following market demands, so it is necessary to carry out quality control and supervision management for all production processes. Quality control and supervision must be carried out from the beginning of the production process to distribution channels to increase consumer confidence, improve product safety assurance, prevent many damaged products, and prevent wasted costs due to losses incurred (Junais et al., 2018).
Quality control of tapioca flour must be considered because if the tapioca flour is of good quality, the processed products produced by consumers will also get good results which will then have the effect of purchasing tapioca flour on an ongoing basis or continuously. In this study, tapioca flour's quality was examined and compared with the quality standards of tapioca flour, namely SNI 3451-2011, PerKBPOM no 36 of 2013, and the standards set by PT Sinar Pematang Mulia. Tapioca flour that does not meet the specified standards must be appropriately handled. This is very important to achieve a good and safe product for consumers. ISSN: 2715-6214 (Print)

RESEARCH METHOD
The process of observing the quality of tapioca flour products at PT Sinar Pematang Mulia II was carried out for seven days with several testing parameters, namely water content, pH, retain sample 100 mesh, impurities mesh 325, SO2, ash content, starch content, whiteness, acid degree, and viscosity. This research was conducted for one month at PT Sinar Pematang Mulia II, Bandar Mataram District, Central Lampung. They collected data in this study through interview observations and literature study. The method of analysis carried out is to compare the results of the analysis using the standard quality requirements of SNI tapioca flour, PerKBPOM No. 36 of 2013, and the standards set by PT Sinar Pematang Mulia.

RESULT AND DISCUSSION
According to the International Standardization Organization (ISO), to obtain quality standardization, a company must cooperate with the parties concerned through consulting experience, technology, and scientific results because the quality standards owned by an industry greatly affect the company's reputation, product accountability, and consumer trust. PT Sinar Pematang Mulia II carries out quality control to ensure the quality of tapioca flour for 24 hours. By conducting physical and chemical analysis and applying quality standards for tapioca flour products that refer to SNI 3451: 2011, PerKBPOM no 36 of 2013, and separate quality standards requested by consumers.
Data analysis was conducted to analyze the causes of the problem. Based on the formulation of the problem above, data was obtained during the implementation of the Practical Work at PT Sinar Pematang Mulia II, and the results can be analyzed. Problem-solving analysis on this problem is carried out by comparing the test results with the standards set by PT Sinar Pematang Mulia II. according to established standards.
Quality Control carries out quality control of Tapioca Flour at PT Sinar Pematang Mulia II based on comparing test results with Standards. The role of Quality Control is very influential on the quality of tapioca flour produced. Its main task is to monitor every production process, and if there is a problem with the quality of tapioca flour, you can directly contact the production staff for follow-up. The parameters used to determine the quality of the final product of tapioca flour are Water Content Test, pH, Retained On 100 mesh, Impurities 325 mesh, SO2, Ash Content, Starch Content, Acid Degree, Whiteness, and Viscosity. The tapioca flour samples were composite samples from 3 shifts (shift 1, shift 2, and shift 3).  Table I. According to research (Andarwulan et al., 2011), excess moisture content can cause a shorter shelf life of tapioca flour, and tapioca flour the higher the humidity and the more likely it is for mold to grow. The presence of water content in foodstuffs is often associated with food quality because it is used as a determinant of the stability index during storage. Food stability and quality are directly affected by water content (Sundari et al., 2015).
Analysis of pH determination for seven days obtained the results of 6.08, 5.62, 5.72, 5.71, 5.69, 5.76, and 5.08. Based on the results obtained, the pH value is still within safe limits. PT Sinar Pematang Mulia II has its pH standard to produce tapioca flour following the criteria desired by consumers; the pH ranges from 4.0 -7.0, as shown in Table 1. In the Indonesian National Standard (SNI 3451:2011), tapioca flour's pH value is not required. However, several institutions require that the pH value of tapioca flour is one of the factors to determine the quality of tapioca flour related to the processing process, namely the process of forming pasta (Pratama et al., 2018). Optimum gel formation occurs at a pH between 4-7 (Whistler et al., 1984).
Analysis of retained on 100 mesh for seven days obtained results of 0.89%, 0.96%, 0.71%, 1.00%, 0.90%, 1.38%, and 0.99%. The fineness of tapioca flour is also very important in determining the quality of tapioca flour. Tapioca flour of good quality is flour that does not clot and has a fineness between 90-100 mesh, although the SNI does not require the fineness of tapioca flour. PT Sinar Pematang Mulia II has its fineness standard to produce tapioca flour following the criteria desired by consumers and standards. The method obtains a minimum of 95% fine flour from the ingredients (the maximum coarse flour yield is 5% from the ingredients). Based on the results obtained, the retained 100 mesh test results are still within safe limits and following factory standards; the yield is less than 5%, as shown in Table I. According to research (Nurani et al., 2013), the coarser the flour particles, the lower the flour. Oil absorption rate. This can be caused by the coarser the flour particles or, the narrower the surface area of the particles, the less possibility of water that can be absorbed by the ingredients during dough making so that the product has a more porous and porous structure, less oil can be absorbed and trapped inside.
Analysis of the 325 mesh residue for seven days obtained the results of 0.17, 0.17, 0.14, 0.22, 0.15, 0.17, 0.14. PT Sinar Pematang Mulia II has its residue analysis standard to produce tapioca flour according to the criteria desired by consumers. The standard is to obtain refined tapioca flour with a maximum residue of 1%, as shown in Table I. Based on the results obtained, the results of the mesh residue analysis are still within safe limits and following factory standards; namely, the result is less than 1%. The analysis of the 325 mesh residue needs to be controlled because it will affect the fineness of the flour produced. According to (Nurani et al., 2013), the varying fineness of flour particles can be caused by differences in the processing process. In the processing of tapioca flour and corn starch, the flour product is obtained through the stages of the starch extraction process from the liquid by precipitation. The starch extract in the form of starch granules has a very fine particle size.
SO2 analysis obtained a result that is 1.28. Based on the results obtained, the SO2 value is still within the safe limits when compared to the quality standard of tapioca flour products PerKBPOM no 36 of 2013 with a maximum SO2 level of 70 ppm can be seen in Table I. SO2 analysis was carried out to determine the SO2 content of tapioca flour was within the safe limit or not. The form of SO2 used is sulfur water. The added sulfur water serves as a preservative and maintains the whiteness of the resulting flour. According to research (Saputro, 2018), if the addition of sulfur is not appropriate, it is not following the Standard Operating Procedure (SOP) from the factory, it will have an impact on consumer health if consumed in the long term and smell produced will smell sulfur (SO2) or typical tapioca flour. If tapioca flour produced after the production process still smells (SO2), it can be ascertained that the mixture (SO2) added as tapioca flour bleach is not following the predetermined dose.
The analysis of ash content was carried out for seven days, and the results obtained were 0.09%, 0.10%, 0.11%, 0.09%, 0.11%, 0.09%, and 0.09%. Based on the results obtained, it can be concluded that the ash content of tapioca flour has met the quality standard of tapioca flour products SNI 3451:2011 with a maximum ash content of 0.5% can be seen in Table 1. According to (Hamsah, 2013), the ash content of a food ingredient indicates the number of minerals contained in the food. Ash content describes the number of minerals that do not burn into volatile substances. Most food ingredients, namely 96%, consist of organic matter and water, and the rest consists of mineral elements, also known as inorganic substances or ash content. Ash content is mineral elements as a residue left after the material is burned until it is free of carbon. The higher the ash content, the worse the flour quality; the lower the ash content, the better the flour quality.
Starch content was analyzed using the starch hydrolysis method by breaking the starch polymer chain into dextrose units. The test was carried out for seven days and the results obtained were 86.86%, 86.87%, 86.78%, 86.87%, 86.88%, 86.89%, and 86.88%. Based on the results obtained, it can be concluded that the starch content of tapioca flour has met the quality standard of tapioca flour products SNI 3451:2011 with a minimum starch content of 75% seen in Table I. According to research (Wijana et al., 2009), the difference in starch content can be caused by various things: differences in the harvesting age of cassava, the type or variety of cassava planted, and technology for processing cassava into cassava. Cassava harvested at a relatively young age of fewer than seven months has lower starch content, while cassava harvested at planting age between 7-9 months will produce cassava with high starch content.
The whiteness analysis was carried out for seven days, and the results obtained were the color of pure white tapioca flour with values of 92.0%, 92.3%, 92.7%, 92.2%, 92.5%, 92.3%, and 93.0%. Based on the results obtained, it can be concluded that the whiteness of tapioca flour has met the quality standard of tapioca flour products SNI 3451:2011 with a minimum whiteness of 91% can be seen in Table I. The flour oven process also influences the color of tapioca flour. According to research (Ega & Lopulalan, 2015), it can be seen that heating HMT sago starch at a temperature of 100 o C gives a lower whiteness value when compared to a heating temperature of 90 o C. The increase in whiteness value in HMT sago starch by heating treatment at 90 o C is thought to be influenced by low pH value, sago starch with heating treatment. This is usually also caused by the browning process on materials that contain carbohydrates. The high drying temperature causes the color of the flour to become darker due to the browning reaction. The quality of tapioca is determined by several factors, one of which is the white color of good tapioca flour (Mustafa, 2016).
The degree of acidity is the dissolution of the organic acids in the sample using a certain organic solvent (95% alcohol) followed by titration with a base (NaOH). Acid degree analysis conducted for seven days obtained the same result, 0.1. Based on the results obtained, it can be concluded that the degree of tapioca flour has met the quality standard of tapioca flour products SNI 3451:2011 with a maximum degree of acidity is 4% in Table 1. According to research (Suismono et al., 1989), the acidity of tapioca flour is likely. This is caused by the content of HCN, organic acids, and CO2 due to starch reshuffle and fermentation.
Viscosity analysis was carried out for seven days; the results obtained were 2500 cp, 2800 cp, 2600 cp, 2700 cp, 2700 cp, 2700 cp, and 2600 cp. Based on the results obtained, it can be concluded that the tapioca viscosity has met the quality standards of PT Sinar Pematang Mulia tapioca flour products with a minimum viscosity of 1700 cp, which can be seen in Table 1. According to research (Ega & Lopulalan, 2015), it can be seen that the higher the temperature, the higher the temperature. When heating is carried out, the faster the sago starch becomes viscous and the lower its resistance to flow (viscosity), and vice versa, the lower the heating temperature, the longer the sago starch becomes viscous, meaning the higher the flow rate (viscosity). The quality of tapioca is determined by several factors, one of which is a high viscosity (viscosity) so that the adhesive power of tapioca remains high (Mustafa, 2016).

CONCLUSION
The results of research and analysis concluded that the quality parameters of PT Sinar Pematang Mulia tapioca flour were water content, pH, flour fineness, residue or impurities, SO2 content, ash content, ISSN: 2715-6214 (Print)


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

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Solanum galapagense ‐derived purple tomato fruit color is conferred by novel alleles of the anthocyanin fruit and atroviolacium loci

Abstract One hypothesis for the origin of endemic species of tomato on the Galápagos islands postulates a hybridization of Solanum pimpinellifolium and Solanum habrochaites . Solanum galapagense accession LA1141 has purple fruit pigmentation, previously described in green‐fruited wild tomatoes such as S. habrochaites or Solanum chilense . Characterization of LA1141 derived purple pigmentation provides a test of the hybridization hypothesis. Purple pigmentation was recovered in progenies derived from LA1141, and the anthocyanins malvidin 3(coumaroyl)rutinoside‐5‐glucoside, petunidin 3‐(coumaroyl) rutinoside‐5‐glucoside, and petunidin 3‐(caffeoyl)rutinoside‐5‐glucoside were abundant. Fruit color was evaluated in an introgression population, and three quantitative trait loci (QTLs) were mapped and validated in subsequent populations. The loci atroviolacium on chromosome 7, Anthocyanin fruit on chromosome 10, and uniform ripening also on chromosome 10 underly these QTLs. Sequence analysis suggested that the LA1141 alleles of Aft and atv are unique relative to those previously described from S. chilense accession LA0458 and Solanum cheesmaniae accession LA0434, respectively. Phylogenetic analysis of the LA1141 Aft genomic sequence did not support a green‐fruited origin, and the locus clustered with members of the red‐fruited tomato clade. The LA1141 allele of Aft is not the result of an ancient introgression from the green‐fruited clade and underlies a gain of anthocyanin pigmentation in the red‐fruited clade.

| INTRODUCTION
hypothesized that species of tomato endemic to the Galápagos, Lycopersicon cheesmanii f. minor, now classified as Solanum galapagense, might have resulted from the hybridization of Solanum pimpinellifolium and Solanum habrochaites progenitors. This hypothesis was based on three unique traits found in both S. habrochaites and S.
habrochaites (Rick, 1961). S. galapagense accession LA1141 has purple pigmentation in immature fruit, similar to species in the green-fruited tomato clade, including S. habrochaites. The presence of this fourth trait common to S. galapagense and S. habrochaites suggested that characterizing the chemical and genetic basis of purple fruit derived from LA1141 could provide a test of Rick's, 1961 hypothesis.
The endemic Galápagos tomatoes possess morphological and physiological traits that distinguish them from other wild species.
These traits include orange fruit color at maturity, yellow-green foliage, tiny seed size, seed dormancy, and affinity for dry conditions (Darwin et al., 2003;Rick, 1961). These species can hybridize easily with cultivated tomatoes, making them useful donors of novel alleles (Rick, 1961). Several genes from Galápagos tomatoes have been used in breeding contemporary varieties. An allele of uniform ripening (u) from Solanum cheesmaniae accession LA0428 is responsible for the uniform distribution of light green pigmentation in immature fruits (Rick, 1967). Alleles conferring jointless ( j 2 ) pedicel (Rick, 1956) and arthritic articulation (j 2in ) (Joubert, 1962, available at: https://tgc.ifas. ufl.edu/vol12/Volume12.pdf) have enabled mechanical harvest. S. cheesmaniae accession LA0422 has a recessive allele, anthocyanin gainer (ag 2 ), which results in fruit and foliage lacking anthocyanin at early developmental stages (De Jong et al., 2004;Rick, 1967). Alleles of the Beta (B) locus found in all S. galapagense and S. cheesmaniae accessions confer high β-carotene and the characteristic orange fruit . Alleles of B from LA0317 and LA0166 have been introgressed into cultivated tomatoes (Stommel, 2001).
Additionally, an accession of S. cheesmaniae (LA0428) was described as having immature fruits with a purple color that resemble Solanum peruvianum (Rick, 1967). Identification and analysis of loci that confer purple fruit color may shed light on broader questions about the evolutionary history of the Galápagos tomatoes.
Water-soluble vacuolar pigments called anthocyanins cause purple fruit pigmentation in species of Solanum (Chaves-Silva et al., 2018;Mes et al., 2008;Timberlake & Bridle, 1982). Within the taxonomic framework of tomato (Solanum sect. Lycopersicon) (Peralta et al., 2008), the red-fruited clade corresponds to the group Esclulentum (Pease et al., 2016), which generally lack anthocyanins in the fruit. The green-fruited clade is grouped into Arcanum, Peruvianum, and Hirsutum based on whole-transcriptome concatenated molecular clock phylogeny (Pease et al., 2016). Purple pigmentation is a characteristic found throughout the green-fruited tomato clade. For example, S. habrochaites accession LA1777 has pronounced anthocyanin spots in its fruit (Dal Cin et al., 2009). Additionally, S. peruvianum fruit is purple-tinged with pigment in lines and blotches (Muller, 1940). The chemical basis of purple fruit derived from tomato species in the green-fruited clade is attributed to the anthocyanins petunidin and malvidin (Jones et al., 2003;Mathews et al., 2003;Ooe et al., 2016). Two loci affect the regulation of anthocyanin accumulation in tomato fruit, one on chromosome 7 and a second on chromosome 10.
We conducted experiments to describe the chemical and genetic basis of purple pigmentation in fruit derived from LA1141. Our results suggest that the regulatory mechanism described for accessions from the green-fruited tomato clade also confers pigmentation in LA1141.
However, the LA1141 alleles of Aft and atv are distinct from those previously characterized. Phylogenetic analysis of Aft sequence does not support a green-fruited origin of the LA1141 locus, which suggests that purple fruit pigmentation in this accession results from a loss of function that disrupts atv and a gain of function that restores Aft. Mutations in Aft may have arisen spontaneously in S. galapagense or be derived from an earlier, red-fruited ancestor, plausibly S. pimpinellifolium. These findings fail to support Rick's (1961) hypothesis on the origin of the Galápagos tomatoes.

| Plant materials and growing conditions
An inbred backcross (IBC) population was initiated in 2014 for the simultaneous introduction and characterization of purple pigmented fruit. The IBC population was derived from an initial hybridization of S. galapagense S.C. Darwin (Berry et al., 1991). A single LA1141 Â OH8245 F 1 plant was backcrossed as the female parent to OH8245. BC 1 individuals were then separately backcrossed again with OH8245 as the pollen donor. BC 2 plants were then selfpollinated with single seed descent in alternating greenhouse and field production cycles to create a BC 2 S 3 IBC population composed of 160 inbred backcross lines (IBLs). During these studies, the IBC population was further inbred to BC 2 S 5 . The BC 2 S 3 IBLs SG18-124 ( Figure 1c) and  were selected based on purple pigmentation in the fruit to generate populations for validation of quantitative trait loci (QTLs). The IBLs SG18-124 and SG18-200 were again crossed to OH8245, and the self-pollination of the resulting F 1 plants created populations with F 2 segregation for specific LA1141 chromosomal regions.
Seedling care for greenhouse and field trials followed the same protocol. The 160 BC 2 S 3 IBLs and the SG18-124 and SG18-200 derived F 2 progenies were sown in 288 cell trays with a cell volume of 13 ml. Greenhouse temperatures were set to 27 C during the day and 25 C at night with a 16-h photoperiod. Photosynthetically active radiation (PAR) was supplied by natural sunlight, 1,000-W metal-halide lamps (Multi-Vapor ® GE Lighting, East Cleveland, OH, USA), and 1,000-W high-pressure sodium lamps (Ultra Sun ® Sunlight Supply, Vancouver, WA) with a target radiation threshold of 250 W m À2 natural light or approximately 113 μmol m À2 s À1 PAR. Fertilization was applied using a 20-20-20 fertilizer (20% N, 20% P 2 O 5 , and 20% K 2 O) (Jack's professional All-Purpose Fertilizer, JR Peters INC., Allentown, PA, USA) delivered at a concentration of 1,000 mg L À1 (200 mg L À1 N) twice per week. Plants were irrigated once or twice per day as needed.
IBC and F 2 progenies were evaluated in field trials as single plants. The BC 2 S 3 IBC population was evaluated with 60-cm spacing and 164 plants, including controls. Progenies derived from SG18-124 and SG18-200 were transplanted for greenhouse and field evaluations of pigmentation in the fruit. Plants with three to five expanded leaves were transferred to 3.78-L containers (Hummert, EARTH City, MO) and spaced 30 cm apart on the greenhouse bench. There were 36 F 2 plants evaluated in the greenhouse. The remaining SG18-124 and SG18-200 derived F 2 progenies were evaluated in field trials with 60 cm spacing with a total of 145 plants harvested.

| Tomato fruit color measurements
Three mature green (MG) fruits were randomly selected from each plot and measured at the midpoint between the fruit's shoulder and the blossom end. Fruit Color was measured with a colorimeter (chromameter CR-300; Minolta Camera Co., Ltd., Ramsey, NJ, USA). Values of the red, green, yellow, and blue components of fruit were obtained using the "L*a*b*" CIELAB color space (Commission Internationale de F I G U R E 1 Heritable fruit pigmentation from Solanum galapagense accession LA1141. We determined a role for several candidate genes underlying the Anthocyanin fruit (Aft), atroviolacea (atv), and uniform ripening (u) loci derived from LA1141. Homozygous LA1141 Aft is designated as Aft, homozygous LA1141 atv is designated as atv, and homozygous LA1141 u is designated as u + . Allele notation follows the rules for nomenclature in tomato genetics (Barton et al., 1955). (a) LA1141 mature green fruit (Aft; atv; u + ). (b) Inbred backcross line (IBL) atv + ;u). (c) IBL SG18-124 (Aft; atv; u + ). (d) IBL SG18-251 (Aft + ; atv + ; u + ) lʼEclairage, 1978). The L* coordinate represented a measure of the darkness or lightness. Coordinates, a* and b*, are measured color along the axis of a color wheel with +a* in the red direction, and -a* in the green direction, +b* in the yellow direction, and -b* in the blue direction (Kabelka et al., 2004). Chroma and hue were derived from measurements of a* and b*. Chroma was calculated as (a* 2 + b* 2 ) 1/2 and used to measure how bright or dull the color was. Hue was calculated using the equation (180/π) [cos À1 (a*/chroma)] for positive values of a*. For negative values of a*, we calculated hue using the equation 360-(180/π) [cos-1 (a*/chroma)] (Darrigues et al., 2008;Kabelka et al., 2004). The average hue, chroma, and L* values were used as the response variable for our genetic studies.

| Anthocyanidin extraction, analysis, and identification
Tomato fruit samples at different stages of maturity from SG18-124 Â OH8245 derived F 2 plants were blended, and 3.5 g of juice was extracted with 4 ml 1% HCl in MeOH. The extracts were dried under nitrogen gas. Anthocyanins were separated using an C18 column (HSS T3, 2.1 Â 100 mm, 1.8 um, Agilent Technologies) and a gradient of water (A) and acetonitrile (B), both with 5% formic acid.
The gradient was as follows: isocratic with 0% B from 0-2 min, linear gradient to 30% B from 2-8 min, linear gradient to 100% from 8-12 min, hold at 100% B for 1 min, and return to initial conditions. Samples were run on an Agilent 1290 ultra high performance liquid chromatography (UHPLC) with photodiode array (PDA) detection, coupled to a high resolution 6545 quadrupole time-of-flight mass spectrometer (QTOF-MS) (Agilent, Santa Clara, CA, USA). The MS was run using electrospray ionization and operated in both positive and negative modes using reference masses for accurate mass determination.

| DNA isolation and genotyping
Genomic DNA was isolated from fresh, young leaf tissue from the 160 BC 2 S 3 progenies, 96 of each F 2 population, and parental lines using a modified CTAB method consistent with previous studies (Sim et al., 2015). Single-nucleotide polymorphisms (SNPs) between OH8245 and LA1141 were identified using a 384-marker panel (Bernal et al., 2020). Genotyping of the BC 2 S 3 progenies was performed using the PlexSeq™ platform as a service (Agriplex Genomics, Cleveland, OH, USA) to detect specific SNPs through a pooled amplicon sequencing strategy.
2.5 | Marker development for candidate genes Selected SNP markers and candidate genes were converted to polymerase chain reaction (PCR) based insertion/deletion (INDEL) markers for visualization on agarose gels. These markers, when appropriate, were added to the linkage maps described below. The genetic notation used in this study followed the rules for nomenclature as recommended (Barton et al., 1955). A summary containing forward and reverse primers, genome location, and expected polymorphism for these markers is available at https://doi.org/10.5281/zenodo. 5650150 (Fenstemaker et al., 2021a). Candidate genes included MYB transcription factor sequences corresponding to atv (MF197509,  Fosberg, 1987in [Fosberg FR (1987b formerly Lycopersicon cheesmaniae L. Riley, 1925in [Riley LAM (1925c] accession LA0483, S. galapagense accession LA1401 and LA1044, and S. lycopersicum variety Indigo Rose. Primers were designed using Primer3 (v..4.0) (Untergasser et al., 2012). These primers were used to genotype LA1141, OH8245, the BC 2 S 3 IBC population, and the subsequent F 2 progenies derived from IBL selections SG18-124 and SG18-200.
PCR was carried out with an initial incubation at 94 C for 3 min, followed by 40 cycles of denaturation at 94 C for 30 s, annealing at 52 C for 30 s, and extension at 72 C for 60 s. After completing the cycles, a final elongation step at 72 C was carried out for 7 min. The PCR products for markers detected as CAPS were digested with a restriction enzyme (Fenstemaker et al., 2021a) for 2 h at 37 C. The PCR products were separated on a 2.5% agarose gel.

| Linkage map construction
A genetic linkage map was constructed based on the IBC population.
The R/qtl package version 1.47-9 was used in the R statistical software environment version 4.03 (Broman et al., 2003;R Core Team, 2020). We used the "read.cross" function from BC s F t tools to read in data, with s = 2 and t = 3 (Shannon et al., 2013). Of the 384 SNPs in the marker panel, 157 were polymorphic in the IBC population, and no markers were removed. A summary of the 157 polymorphic SNPs is available at https://doi.org/10.5281/zenodo.5650152 (Fenstemaker et al., 2021b). The genetic map was constructed by using the "est.map" function in R/qtl. Markers were placed in the same linkage group if they had a logarithm of the odds (LOD) score greater than 1.8 and an estimated recombination fraction lower than .45. The Kosambi map function was used for map construction and to convert recombination frequency to genetic distance (Kosambi, 1944). The marker order in each linkage group was estimated with the functions "orderMarkers" and "ripple"in R/qtl. Changes in chromosome length and loglikelihood were investigated, dropping one marker at a time with the function "droponemarker" in R/qtl. Marker order was compared with the physical position in the Tomato Genome version SL4.0 (Hosmani et al., 2019) using both linear (adjusted correlation coefficient R 2 ) and rank regression (rho(ρ)) to assess linkage map quality.

| QTL analysis in BC 2 S 3 IBLs
Composite interval mapping (CIM) was used for QTL detection (Zeng, 1994) using the "cim" function in the R/qtl package (Broman et al., 2003). Analysis was performed using a 2-cM step, one marker selected as a cofactor, and a 40-cM window with cofactor and window selected due to limited recombination and expected skewed segregation in the BC 2 S 3 population. Haley Knott regression (Haley & Knott, 1992) (hk) was chosen as the solution-generating algorithm.
Significance thresholds were generated by using the permutation test (α = .05, n = 1,000; Churchill & Doerge, 1994). The resampled LOD cutoffs used were LOD = 6.8 for hue, LOD = 4.5 for chroma, and LOD = 3.65 for L*. Genetic effects were evaluated as differences between phenotype averages expressed as regression coefficients using the "fitqtl" function with the argument "get.ests = TRUE" and "dropone = FALSE" in R/qtl. Additionally, the percent phenotypic variance explained was estimated by the "fitqtl" function with the argument "dropone = TRUE" in R/qtl.

| QTL validation
The IBLs SG18-124 and SG18-200 were chosen because of pigmentation in their fruit (Figure 2b,c). Seedlings were also grown as previously described. Segregating SG18-124 Â OH8245 F 2 and SG18-200 Â OH8256 F 2 progenies were transplanted to the field and greenhouse, and fruit pigmentation was measured using the Minolta CR300 colorimeter as described above. Marker data were scored on 91 progenies derived from the SG18-124 Â OH8245 F 2 population and 90 from the SG18-200 Â OH8245 F 2 population. Genetic effects and allele substitutions were evaluated using linear model analysis of variance (ANOVA) as implemented by the "lm" function in the R core package (R Core Team, 2020). The linear model was where Y was the color trait value, μ x was equal to the population mean, M was the effect of each marker allele, and E was the associated error, equivalent to genotype (marker). We compared the marker-locus genotypic classes of LA1141 and OH8245. The markers Ant1_1, An2-like_exon2_intron2, atv_ex4, u_gal_3, and BetaRSAcorresponding to candidate genes of interest were tested (Fenstemaker et al., 2021a). Marker evaluations were conducted in both F 2 populations independently. We used F-tests as previously described to determine if hue, chroma, and L* were associated with significant differences in marker-locus genotypic classes and used the mean phenotypic differences to estimate the effect of allele substitutions.
Additionally, we tested possible allelic combinations of Aft and atv in the IBC and F 2 populations. We used the linear model Y = μ x where Y was the color trait value, μ x was equal to the population mean, M 1 and M 2 were effects of individual marker alleles, M 1 :M 2 was the interaction between marker alleles, and E was the associated error, equivalent to genotype (marker) to test for significant markers interactions. We conducted a linear model ANOVA using the "lm" function in the R core package (R Core Team, 2020) to test the pairwise combination of chromosome 7 (atv) and chromosome 10 (Aft). When marker classes were significantly differ- Tool (BLAST) version/2020-04 (Altschul et al., 1990) program. Our workflow parsed through sequence matches, identified the highest quality match, and created a FASTA output file. Parsing was facilitated by "SearchIO", "Seq", and "SeqIO", functions in BioPerl (Stajich et al., 2002) following implementation of the "blastn" function in the BLAST core package. The steps in the pipeline were automated using the Bash scripting language (Gnu, 2007)  To retrieve CDS from NCBI, we accessed the "RefSeq" section of the Genbank records mentioned above. The CDS were extracted from the "features" section of the Genbank records and exported as FASTA files. The UGENE v. 37 software package (Okonechnikov et al., 2012) was used for sequence trimming prior to alignment using MUSCLE (version 3.8.31) (Edgar, 2004) in the OSC Unix command line.
Phylogenetic trees were constructed using the phangorn R package (Schliep, 2011) for the R2R3 MYB-encoding genes Ant1and An2-like. Genomic sequence files were combined from the MYBencoding genes An2-like and Ant1 to create a single Aft locus contig, aligned in MUSCLE and imported into phangorn. We constructed maximum likelihood (ML) trees based on the nucleotide alignment using the general time reversible model with the rate variation among sites described by a gamma distribution and the proportion of invariable sites (a.k.a. GTR + G + I model). The "optim.pml" function was used to optimize model parameters with a stochastic search algorithm to compute the likelihood of the phylogenetic trees (Nguyen et al., 2015). This methodology was used for both genomic and CDS sequences. Clade support was estimated with 1,000 bootstrap replicates using the function "bootstrap.pml." Phylogenetic studies that used genomic sequence were rooted using S. lycopersicoides as an outgroup. Studies that used CDS were rooted using Arabidopsis as an outgroup. Trees were drawn and annotated using the Interactive Tree Of Life (ITOL) (Letunic & Bork, 2021; available at https://itol. 3.3 | LA1141 Â OH8245 BC 2 S 3 linkage map quality Linkage maps were constructed based on marker data from the BC 2 S 3 IBC population and defined 13 linkage groups corresponding to each tomato chromosome. We split chromosome 1 into two linkage groups (1a and 1b) because of a recombination fraction greater than .45 between adjacent markers. The total number of markers in each linkage group ranged between 2 and 27, and linkage group 4 had the most markers at 27 (Table 1). The centimorgan (cM) length per linkage group ranged between 25.8 and 121.6 cM ( Table 1). The average cM distance between markers was 8.1, and the largest distance in cM between markers was 41.8 (Table 1). SNP marker physical position using the tomato Sl4.0 physical map (Hosmani et al., 2019) agreed with the estimated genetic position using both linear correlation and rank correlation ( Table 1). As previously demonstrated, correlations are not perfectly linear due to reduced recombination in the pericentromeric region (Sim et al., 2012). Linear correlations ranged from .28-.99, while rank correlations ranged from .96 to 1 (Table 1).

| QTL analysis of tomato color in the LA1141 Â OH8245 BC 2 S 3 population
We identified three putative QTLs that explained between 8.24% and 35.53% of the total phenotypic variation for hue, chroma and L* ( Figure 3 and Table 2). All QTLs that contribute to the purple color are derived from the LA1141 donor parent, with purple pigmentation defined by an increase in hue and a decrease in both chroma and L* (Table 2). A region on the distal arm of chromosome 10 explained between 22.63% and 24.04% of the total phenotypic variation and increased hue between 6.74 and 7.5 ( Figure 3 and Table 2). Two QTLs, one on the proximal arm and one on the distal arm of chromosome 10, were associated with chroma and explained between 18.02% and 28.53% (proximal arm) and between 15.95% and 23.08% (distal arm) of the total phenotypic variance ( Figure 3 and Table 2).
Two QTLs were associated with L*, one on chromosome 6 and one on chromosome 10 (proximal) (Figure 3 and Table 2). These QTLs explained between 8.24% and 35.53% of the total phenotypic variation ( Table 2). The QTL on chromosome 10 explained between 22.03% and 35.53% of the phenotypic variation and reduced L* by 9.23 units ( Table 2). The QTL on chromosome 6 explained between 8.24% and 10.13% of the phenotypic variation and reduced L* between 4.53 and 5.05 units (Table 2).

| Candidate genes
Candidate genes were selected because of their previously characterized role in regulating tomato fruit pigmentation and because of their locations within the physical intervals of our QTLs ( Table 2). The

2018) because of its previously described synergistic interaction with
Aft which results in a purple phenotype similar to what we observe in our deep purple accession (Figure 1c). We added these markers to the linkage maps described above and used them in our QTL analysis.
3.6 | QTL mapping using candidate genes in the IBC population  Table 2).
Regions on chromosome 6 and the proximal arm of chromosome 10 were targeted for measurements of L*. The marker u (LOD = 15.25) exceeded our resampled LOD cutoff (LOD = 3.65), explained 35.53% of the total phenotypic variance, and reduced L* by 9.32 units ( Table 2). The marker B (LOD = 1.26) did not exceed our resampled LOD cutoff for L*, and our QTL analysis failed to support a role for B as a candidate gene on chromosome 6. Additionally, the Aft markers (LOD = 1.13) and atv (LOD = 1.48) did not appear to be associated with L* (Table 2).
Although the marker atv did not exceed our LOD significance thresholds for hue, chroma, or L* (  deep purple phenotype in the BC 2 S 3 progenies suggested two unlinked loci were responsible. The known regulatory mechanism involving MYB-encoding genes underlying atv and Aft led us to test for interaction effects for loci on chromosome 7 and chromosome 10. The interaction between LA1141 Aft and the LA1141 atv was significant (p = < 2.2e-16) (Figure 4). We compared the hue values of BC 2 S 3 IBL progenies with Aft and atv derived from LA1141 (Aft;atv) to those derived from OH8245 (Aft + ;atv + ) (Figure 4a). The BC 2 S 3 IBLs with both the LA1141 Aft and atv had higher hue values than the Aft + ; atv + genotypes ( Figure 4a). Additionally, we compared all possible allele combinations, including the genotypes Aft;atv + and and Aft + ; atv. The Aft;atv LA1141 genotype had higher hue values than all other combinations ( Figure 4a). However, the Aft;atv + genotype had higher hue values than the Aft + ;atv and Aft + ;atv + genotypes (Figure 4a).

| Confirmation of QTLs in the F 2 validation populations
We evaluated F 2 populations originating from the selected IBL proge- In the SG18-124 derived F 2 population, the markers Ant1 (p = 1.513e-09) and An2-like (p = 2.118e-09) were significantly associated with hue, explained 37% of the phenotypic variation, and increased hue by 19.45 and 22.05 , respectively (Table 3). The marker atv (p = .022) was also significantly associated with hue, explained 9% of the phenotypic variation, and increased hue by 11.99 (Table 3). The marker u (p = .901) was not significant for hue (Table 3). However, the marker u (p = 2.071e-05) was significantly associated with chroma, explained 23% of the phenotypic variation, and decreased chroma by 10.67 units ( Table 3). The markers An2-like (p = 4.051e-04) and Ant1 were significantly associated with chroma, explained 14% and 27% of the total phenotypic variation, and decreased chroma by 10.80 and 12.23 units ( Table 3). The marker u (p = 3.181e-04) was significantly associated with L*, explained 17% of the phenotypic variation, and decreased L* by 10.38 units ( Table 3).
In the SG18-200 derived F 2 population, the markers atv and u were monomorphic (Table 3). Therefore, we did not test the estimated effects of allele substitutions and associations in this population. The markers Ant1 (p = 5.702e-04) and An2-like (p = 3.691e-05) were significantly associated with hue, explained 17% and 23% of the phenotypic variation, and increased hue by 4.36 and 5.03 (Table 3).
We validated the interaction between homozygous LA1141 Aft and homozygous LA1141 atv in the F 2 progeny (Figure 4b). Our results confirm that an interaction between Aft and atv is needed for the deep purple fruit phenotype (Figure 1c),  Color was measured as hue, chroma, and L* in the OH8245 Â LA1141 BC 2 S 3 population. b Logarithm of the odd (LOD) significance cutoffs were determined by a resampling of the data (α = .05, N = 1,000 permutations). LOD cutoffs for traits were hue (LOD = 6.8), chroma (LOD = 4.5) and L* (LOD = 3.65).
c Genetic effects were evaluated as differences between phenotype averages expressed as regression coefficients. d Percent variance explained was estimated by 1-10 À2 LOD/n , where n is the sample size and LOD is the LOD score. e Physical position in base pairs corresponds to the Tomato Genome version SL4.0 (Hosmani et al., 2019).
for Ant1 (Fenstemaker et al., 2021d. There were several unique SNPs and INDELs in the LA1141 An2-like sequence, but none of them were in the conserved R2R3 domains (Fenstemaker et al., 2021d). However, LA1141 possesses the previously characterized G to A SNP in the 5 0 splice site of the second intron (Sun et al., 2020;Yan et al., 2020;Fenstemaker et al., 2021d). Sequencing reads covered 1,182 of 1,012 of LA1141 and 1,012 out of 1,012 bps of OH8245 from the first putative start codon in the R2R3 MYBencoding gene Ant1. In the third exon of the LA1141 Ant1 sequence, there is 170-bp insertion/deletion (INDEL), which contained MYB core type 1 and type 2 cis-regulatory elements, an AC-rich sequence type 2 cis-regulatory element (Fenstemaker et al., 2021e). Sequence analysis suggests that LA1141 may have a functional R2R3 MYB activator at Aft and the R3 MYB repressor corresponding to atv is likely nonfunctional. Additional characterization of transcripts, proteins, and protein interactions is needed for An2-like, Ant, and atv to confirm functional changes.
thaliana MYB genes that were determined to be homologous to Solanum Aft sequence were used as an outgroup to root the tree (supporting information Figure S1). The ML phylogeny separated Ant1 and An2-like CDS with 98.4% bootstrap support (supporting information Figure S1). A. thaliana and S. miltiorrhiza clustered closer together compared with accessions of Solanum for both Ant1 and An2-like (supporting information Figure S1). For Ant1 CDS, accession LA1141 clustered with members of the red-fruited clade with 81.3% bootstrap support. For An2-like CDS, accession LA1141 clustered with members of the red-fruited clade with 49.7% bootstrap support (supporting information Figure S1). Fruit color was measured as hue, chroma, and L* in the BC 2 S 3 IBL derived F 2 populations. b ANOVAs were conducted, and F-tests were used to determine if significant variation in hue, chroma, and L* was associated with differences in markerlocus genotypic classes. If NA, the marker was not segregating in the population and therefore could not be tested for differences in marker-locus genotypic classes. c F-tests to determine if hue, chroma, and L* were associated with significant differences in marker-locus genotypic classes and used the line mean differences to estimate the effect of allele substitutions. d Adjusted correlation coefficient (R 2 ) calculated from linear model analysis of variance (ANOVA) is the percent of total phenotypic variance explained. e Physical position in base pairs corresponds to the Tomato Genome version SL4.0 (Hosmani et al., 2019).
F I G U R E 5 Sequence polymorphisms found in genomic regions of Solyc07g052490 (SlMYBatv) underlying the atroviolacium (atv) locus.
Exons are highlighted green, and introns are unhighlighted black text. Exon and intron boundaries are consistent with previous studies (Cao et al., 2017;Colanero et al., 2018). Start and stop codons are highlighted yellow. Single-nucleotide polymorphisms (SNPs) and other sequence variants are highlighted gray with red text. The causal 4 base pair (bp) INDEL (slmybatv) previously characterized in the tomato cultivar Indigo Rose (IndigoR) (Cao et al., 2017) is shown at bp position 681. Two G to A SNPs located at bp positions 694 and 696 are present in a region identified by CRISPR/CAS9 as important for the function of the conserved R3 domain (Yan et al., 2020). Sequences were aligned with ClustalW (Larkin et al., 2007)  Tomato color depends on the type and quantity of pigments synthesized in the fruits. Anthocyanins are responsible for the purple coloration of immature LA1141 fruit. Delphinidin-3-rutinoside and petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside were the major anthocyanins identified. As fruit ripened, the predominant anthocyanin changed from petunidin 3-(coumaroyl)rutinoside-5-glucoside in the MG stage to malvidin 3(coumaroyl)rutinoside-5-glucoside in the breaker stage ( Figure 2). The chemical basis of pigmentation in progenies derived from LA1141 is consistent with those identified in introgression lines containing alleles from the green-fruited wild relatives (Jones et al., 2003). Phenotyping with quantitative measurements of color allowed us to distinguish classes of fruit that were useful for later genetic analysis. Cao et al. (2017) reported that it was difficult to distinguish marker classes of atv with qualitative phenotyping, but we were able to detect differences in hue values between homozygous and heterozygous genotypes ( Figure 4b). Additionally, our linkage analysis using quantitative measurements distinguished classes and showed that Aft is necessary to recover light purple color in progenies underlying the uniform ripening (u) locus maps to the proximal arm and mediates the brightness or dullness of the color. Accession LA1141 has a functional Golden 2-like allele underlying the u locus. The u locus increases chromoplast number, chlorophyll accumulation, and changing chromoplast distribution (Powell et al., 2012). This chlorophyll accumulation causes immature fruit to have patches of darker green color, especially on the fruit shoulders ( Figure 1d). Sequence analysis of MYB-encoding genes underlying the Aft locus suggested that LA1141 may have a functional R2R3 MYB activator which could explain its purple pigmentation in early stages of fruit development, as measured by hue, chroma, and L*. A nonfunctional allele of atv on chromosome 7 was detected based on interactions with Aft that increased pigmentation measured as hue (Figure 4a). The QTLs and the interaction between chromosomes 7 and 10 were also validated in the subsequent IBL derived F 2 generations (Figure 4b).
Two QTLs were associated with L*, one on chromosome 6 and one on the proximal arm of chromosome 10. Only the QTL on chromosome 10 was validated in subsequent generations (Table 3). The region on chromosome 10 mapped to u. The u locus is likely affecting measurements of fruit darkness for similar reasons mentioned above.
We expected the QTL on chromosome 6 to be associated with the Beta locus. However, mapping B failed to support this locus as a candidate (Table 2). We could not identify a candidate for the QTL on chromosome 6 corresponding to L* in the IBC population. However, the QTL on chromosome 6 only explained 10% of the phenotypic variance compared with 35% of the variance explained by u (Table 2).
Additionally, when we mapped B in the subsequent F 2 populations, we could detect association in only one of the populations (Table 3).
In the SG18-200 derived F 2 population, B was associated with hue but not with chroma or L* (Table 3). We believe that our ability to detect B in this population is attributed to the monomorphic alleles for atv and u reducing the range of hue (Table 3).
The wild tomato relative S. chilense is the source of Aft in cultivated tomato varieties such as Indigo Rose (Jones et al., 2003;Mes et al., 2008). Introgression of Aft from accession LA1141 offers an alternative source of this trait and may have advantages such as greater recombination and potentially less linkage drag (Hamlin et al., 2020). Deep purple pigmentation will be enhanced by the addition of atv and may be further increased in nonuniform (u) ripening backgrounds.

| The primary regulatory mechanism for anthocyanin accumulation is conserved in LA1141
The interaction between chromosome 7 (atv) and chromosome 10 (Aft) in the LA1141 Â OH8245 IBC population results in deep purple fruit (Figure 1c). This interaction suggests that the role of synergistic MYB regulatory genes underlying loci on 7 and 10 is conserved between LA1141 and the green-fruited species. A complex of interacting MYB transcription factors, basic helix-loop-helix transcription factors (bHLH), and WD40 repeat domains (WDR), known as the MYB-bHLH-WDR (MBW), modulate anthocyanin accumulation in plants . The R2R3 MYB activators compete with the R3 MYB repressors for interaction with the bHLH transcription factor in the MBW complex . A CRISPR/Cas9 mediated silencing of MYB genes underlying the Aft locus suggested that only An2-like is needed for purple pigmentation in the peel of the tomato variety Indigo Rose (Yan et al., 2020). The same study showed that restoring function of atv in Indigo Rose reverts the coloration back to the light purple phenotype we observed in SG18-200 (Figure 1b) (Yan et al., 2020). Additionally, atv sequence targeted using CRISPR in the coding region of the second exon, where we observed the G to A SNP in LA1141, resulted in a loss of function of the R3/bHLH binding domain in LA1996 (Yan et al., 2020). This targeted mutation caused a purple phenotype similar to what we observed in our deep purple accession (Figure 1c).

| The potential origin of Aft and atv mutations in accession LA1141
In the red-fruited clade/Esculentum group, the structure of the Aft phylogeny places S. galapagense accessions closer to S. pimpinellifolium, S. lycopersicum var. cerasiformae, and cultivated tomatoes, which is consistent with previously published Solanum phylogeny (Pease et al., 2016). Additionally, results from the outgroup rooted tree using CDS from distantly related species suggest that the greenfruited clade may be ancestral as proposed previously (Peralta & Spooner, 2001). Pigmentation in the tomato clade of Solanum is con- It is interesting to speculate about how LA1141 acquired its purple fruit and how selective forces could maintain this pigmentation.
The duplication of MYB transcription factors in flowering plants in general and the locus of linked family members on chromosome 10, specifically, provide opportunities for diversification and selection (Pickersgill, 2018). Our data do not suggest how this mutation is maintained. The occurrence of purple fruit in LA1141 could be indicative of convergent evolution with tomatoes in the green-fruited clade due to unidentified selective pressure. Alternatively, the mutation could be maintained simply by chance. The flower morphology of S.
galapagense is indicative of a strong inbreeding tendency (Rick & Fobes, 1975), and previous studies have suggested that there is limited genetic diversity within the species (Pailles et al., 2017). Consequently, high and low anthocyanin traits may have become fixed in different populations of Galápagos tomatoes because of genetic drift combined with inbreeding. It is also possible that purple pigmentation serves an adaptive role related to the synthesis of protective anthocyanins under high irradiance or the enticement of organisms that disperse seed (Grotewold, 2006). As an example, orange fruit is postulated to have a selective advantage on the Galápagos Islands due to seed disperser color preferences (Gibson et al., 2021). An Investigation of known seed disperser preferences on the Galápagos Islands and LA1141 fruit could elucidate a possible evolutionary mechanism. However, the complete absence of anthocyanins in some populations of Galápagos tomatoes, such as accessions of S.
cheesmaniae from the San Cristobal Island (Rick, 1967), is incompatible with a theory involving selective forces acting on purple color.
Evolutionary hypothesis explaining the maintance of purple pigment in LA1141 must also take into account the role of the atv loss of function mutation on chromosome 7. The sequence data presented here suggest that multiple loss of function alleles at atv occurs on the Galápagos Islands. It is possible that purple pigmentation is not costly for the plant, and there is a lack of selective pressure on the Galápagos Islands to maintain the function of atv:similar to the loss of function of phytoene synthase-1 at the yellow flesh (r) locus resulting in yellow fruit with low carotenoids (Gibson et al., 2021). A lack of selective pressure may explain why atv alleles may be more widespread in Galápagos tomatoes. Hypothesis related to adaptive maintenance, as discussed above, may offer an alternative explanation.

| CONCLUSION
This work describes the chemical and genetic basis of purple pigmentation in the fruit of accession of S. galapagense LA1141. The anthocyanins malvidin and petunidin are responsible for this color. Genes underlying the atv, Aft, and u loci are implicated as candidates for major QTLs. The loci atv and Aft interact, suggesting that the same mechanism producing anthocyanins in other purple fruited tomato accessions is responsible for pigment patterns in LA1141 fruit. Aft was previously known only from wild accessions in the green-fruited clade, and we probed Rick's hypothesis about an ancient hybridization event between progenitors of S. galapagense using genomic sequence from the Aft locus. Our phylogenetic analysis concluded that a functional allele of Aft in LA1141 is not derived from introgression from a green-fruited relative. The LA1141 Aft clusters with other Galápagos accessions and S. pimpinellifollium accessions from the South American mainland. Our findings guide us toward a better understanding of purple color found in the endemic Galápagos tomatoes and provide additional resources for characterizing anthocyanin biosynthesis in wild tomato relatives.

ACKNOWLEDGMENTS
We thank Jihuen Cho and the farm crews from the Ohio Agricultural

CONFLICT OF INTEREST
The authors have no conflict of interests to declare.