Factors That Affect the Microbiological Stability of “Chicha Morada” During Its Production on an Industrial Scale --Manuscript Draft

Journal of Food Protection

Factors that affect the microbiological stability of “Chicha Morada” during its production on an industrial scale --Manuscript Draft--

Manuscript Number: JFP-21-190R1 Article Type: Mini-Review Paper Section/Category: Food Microbiology Keywords: Foodborne bacteria; hot-fill process; non-carbonated traditional beverages; purple corn drink; Spoilage microorganisms

Corresponding Author: Félix Giovani Ramos Guerrero Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 Universidad Ricardo Palma Lima, PERU First Author: Félix Giovani Ramos Guerrero Order of Authors: Félix Giovani Ramos Guerrero Benedicta C. López Flores Juan C. Ramos Gorbeña Marcial I. Silva Jaimes Manuscript Region of Origin: PERU Abstract: “Chicha Morada” also known as purple corn drink (PCD), is a traditional non- carbonated beverage commonly prepared at homes and restaurants in Peru. However, in recent years, it is being produced at an industrial scale aiming to extend its shelf-life, expand its marketing and make it known worldwide. Traditionally, this beverage, main component being purple corn (Zea mays L.), was made and consumed quickly, and in some cases, stored under refrigeration until consumption, but never beyond 24 to 48 hours. With its industrialization, factories are presented with challenges to design and provide adequate protection of the beverage, assuring its quality and safety. Although its production at an industrial level is very similar to other non-carbonated drinks containing fruit juice, several processing factors could affect the microbiological stability desired for this beverage, such as the storage of the purple corn drink extract. In this document, a critical review of the production process (raw materials, production stages and forms of commercialization) that can have a direct impact on the contamination of the beverage was made. Recommendations are made for improving the control points in the industrial process and to avoid potential microbiological problems.

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Received: April 30, 2021; Accepted: August 4, 2021; Published Online Early: August 2021

Félix G. Ramos Guerrero, Benedicta C. López Flores, Juan C. Ramos Gorbeña, and Marcial I. Silva Jaimes. 2021. Factors that affect the microbiological stability of “Chicha Morada” during its production on an industrial scale. In Press. https://doi.org/10.4315/JFP-21-190

This Online Early paper will appear in its final typeset version in a future issue of the Journal of Food Protection. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

Mini-Review Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021

Running title: Microbiological aspects of industrialized “Chicha Morada”

Factors that affect the microbiological stability of “Chicha Morada” during its production on an

industrial scale

Félix G. Ramos Guerrero1,2, Benedicta C. López Flores2, Juan C. Ramos Gorbeña1, and Marcial I.

Silva Jaimes1,3

1 Instituto de Control y Certificación de la Calidad e Inocuidad Alimentaria (ICCCIA), Universidad

Ricardo Palma, Av. Benavides 5440. Urb. Las Gardenias, Lima 33, Perú.

2 Centro Latinoamericano de Enseñanza e Investigación de Bacteriología Alimentaria (CLEIBA), Facultad

de Farmacia y Bioquímica, Universidad Nacional Mayor de San Marcos, Jr. Puno 1002, Lima 1, Perú.

3 Departamento de Ingeniería de Alimentos y Productos Agropecuarios, Facultad de Industrias

Alimentarias, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 12, Perú.

Key words: Foodborne bacteria, hot-fill process, non-carbonated traditional beverages, purple corn drink,

spoilage microorganisms

 Author for correspondence. Tel. +51 989253990; Fax: +1 2059630032; E-mail: [email protected]

ABSTRACT

“Chicha Morada” also known as purple corn drink (PCD), is a traditional non-carbonated beverage commonly prepared at homes and restaurants in Peru. However, in recent years, it is being produced at an industrial scale aiming to extend its shelf-life, expand its marketing and make it known worldwide. Traditionally, this beverage, main component being purple corn (Zea mays L.), was made and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 consumed quickly, and in some cases, stored under refrigeration until consumption, but never beyond 24 to 48 hours. With its industrialization, factories are presented with challenges to design and provide adequate protection of the beverage, assuring its quality and safety. Although its production at an industrial level is very similar to other non-carbonated drinks containing fruit juice, several processing factors could affect the microbiological stability desired for this beverage, such as the storage of the purple corn drink extract. In this document, a critical review of the production process (raw materials, production stages and forms of commercialization) that can have a direct impact on the contamination of the beverage was made. Recommendations are made for improving the control points in the industrial process and to avoid potential microbiological problems.

HIGHLIGHTS

 PCD is likely to be re-contaminated after pasteurization step.

 Microbiologic control of cooling water can avoid re-contamination.

 Yeasts represent the main group of microorganisms that can cause spoilage in PCD.

 If present in PCD, pathogenic bacteria is not likely to survival. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021

Peru is a South American country known for its farm products and renowned gastronomy. The country is a popular tourist destination due to availability of a variety of its traditional dishes and regional drinks (43). Among the different cereals that are cultivated in Peru since ancient times, there is a particular variety of corn that has an intense purple coloration in the plant, corncob and pericarp of its grains (34).

This particular color is due to the presence of anthocyanins (cyaniding-3-glucoside, pelargonidin-3-

glucoside and peonidin-3-glucoside) and is considered as a bioactive compound with antioxidant capacity Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021

(23, 71, 72).

Purple corn (Zea mays L. amilaceae st.) is mainly produced in 8 departments of Peru (Lima,

Huánuco, Ancash, La Libertad, Arequipa, Ayacucho, Ica and Cajamarca) with its sowing season being from August to October (Peruvian Highlands) and from April to September (Peruvian coast). From 2011 to 2015, purple corn was nationally produced at an annual growth rate of 25% (46). The type of purple corn most used, and commercialized, in Lima is “Morado Canteño”, a native variety of the north-central department of Lima that grows between 500 and 2400 m above sea level. However, there are other adapted varieties, such as “Morado Caraz”, “Morado Arequipeño”, “Morado Cusco” and “Negro de

Junín” (46). Varieties improved by the National University of Agriculture La Molina include PMV-581 and PMV-582 (34), and the INIA 601, developed by the National Institute of Agricultural Innovation, a variety that stands out for its higher anthocyanin content and grain yield in comparison with other varieties

(37).

Purple corn drink (PCD), commonly known as “Chicha Morada”, is a non-carbonated Peruvian beverage manufactured mainly from the cooking of purple corn in water with fruits and spices. Unlike other Latin-American regional products called “chicha”, such as “chicha de jora” (45, 65) or “chicha de

Guiñapo” (66), this beverage is not fermented. This product is considered a traditional beverage in Peru

(73) because of its purple color characteristics and its particular aspect, flavor and smell, and also represents an important part of the non-alcoholic beverage market. PCD is currently being considered as a

4 possible functional beverage due to the cholesterol-lowering effects of its anthocyanins, as well as antihypertensive and antioxidant properties (2, 3, 23, 29).

This traditional beverage, particularly refreshing, has been prepared in Peruvian homes for decades as the most famous dishes of Peruvian gastronomy and is routinely served at children´s parties and other family gatherings (52). Also, due to its popularity and widespread acceptance among Peruvian Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 consumers, movie theater chains have chosen to include PCD in their beverage menu. In recent years, this ready-to-drink product has begun to be produced on an industrial scale with an extended shelf-life, attractive packaging and is commercialized on a national and international scale, posing new challenges in terms of ensuring its quality and safety.

The aim of this review was to discuss in detail the various factors (raw materials, production stages, and commercialization methods) that can influence the microbiological stability of PCD during production process on an industrial scale. This paper will assist factories involved in the manufacture and marketing of the pasteurized non-carbonated beverage known as "Chicha Morada" in improving their

HACCP systems.

INDUSTRIAL PROCESSING OF “CHICHA MORADA”

Chicha Morada's with main ingredient purple corn is a unique, refreshing drink that contains a variety of nutrients beneficial to the human body (Table 1). The beverage's industrial process was developed simulating the preparation method used in Peruvian homes. The process can be divided into four stages: preparation of purple corn drink extract, standardization, thermal inactivation and filling- packaging. Each step performs a specific function, helping to develop the unique sensory, physical- chemical and microbiological characteristics, as well, giving it the microbiological safety and high quality that distinguishes this drink from others. The production process is depicted in Fig. 1, and each stage of the process is outlined in detail below.

Preparation of purple corn drink extract. The Peruvian beverage process starts with the inspection, sampling and analysis of each raw material, ingredient, and packaging material (primary and secondary) according to standard operating procedures and good manufacturing practices.

Depending on the formulation, ingredients are fractioned to meet the quantity specified in the production worksheet, and then delivered to the production area. Purple corn and fruits are washed and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 brushed using potable water. These steps are fundamental for eliminating dust/dirt adhering to the surface and, consequently, to reduce microbial load. They are then cut up and placed in a tank previously filled with potable water. Appropriate controls are carried out on the water to ensure its quality as well as to consistently meet production specifications.

To date, there is no consensus on the formulation for this drink, which varies primarily in the amount of purple corn and fruits used, as well as the ratio of purple corn to potable water used to obtain the extract. For example, Manrique (34) uses a ratio of 1:6 (kg of purple corn: L of potable water), while

Salvador-Reyes and Clerici (52) use a 1:4 ratio. Spices are incorporated to the formulation which contribute to the sensory characteristics of the extract. It is worth mentioning that the color of the extract is dependent on the quantity of purple corn used in the formulation and the way anthocyanin is extracted and stabilized (12, 21).

Major or minor color in the final extract depends on the boiling time. Temperatures greater than

100 °C are commonly applied for 20 to 60 minutes as a minimum (50, 52, 64). Finally, this product is filtered to separate the cooked purple corn, fruits and spices from the purple liquid, called “purple corn drink extract”. Some formulations consider reusing these cooked ingredients by incorporating potable water and cooking again, resulting in a second extract that is mixed with the one already obtained. In some cases, purple corn is boiled in water without the addition of any other ingredients and the resulting product is known as “purple corn extract”. This extract, while still hot, is transferred through pipes to other tanks where it is stored until it is standardized with the rest of the ingredients.

Standardization. This part of the process involves two stages: blending and homogenization. In the blending step, sugar or sweeteners, acids, preservatives, and other ingredients are incorporated into the purple corn drink extract. Depending on the formulation, a certain amount of sugar is added to achieve a minimum of 10.0 °Brix (60); however, this is not mandatory since there are several formulations that currently contain less sugar (less than 6.0 °Brix) due to health concerns and applicable regulations. Lemon

juice or citric acid are commonly used in the recipe to acidify the beverage (lowering the pH typically to Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 around 3.0), as well as to create a hurdle against pathogenic bacteria. These additives improve the taste of the final beverage, making it a refreshing drink. Preservatives such as sorbates and/or benzoates may or may not be included in the beverage’s formulation; if incorporated, these additives extend the shelf-life of the product and create a hurdle effect against the main spoilage microorganisms (aciduric bacteria, mold and yeasts).

Next, the homogenization step produces a uniform lot, maintaining the same characteristics through the entire filling and packaging process. In this case, a proper combination of time and stirring rate yields a beverage that meets the physico-chemicals parameters defined for this product. The quality assurance department is responsible for ensuring that all parameters are in accordance with technical specifications before releasing the product for heat treatment.

Thermal inactivation. Pasteurization is a step commonly applied in the industrial scale production of PCD. Significant reductions of microbial load, both spoilage and key pathogenic bacteria are achieved through temperatures and times greater than 71.1 °C for 3 seconds (35), which is similar to treatment applied to industrialized acidic fruit juices. This is the most important processing step in the microbiological stability of the Peruvian beverage, and its compliance is in accordance with several regulations, such as the standard of 5-log pathogen reduction based on the Food and Drug

Administration’s Guidance for Industry: Juice HACCP Hazards and Controls Guidance (63). This thermal treatment can be carried out by using tubular pasteurizers or plate interchanges (dynamic process, continuous) or in batches in a tank (static process, not continuous). In the literature, temperature and time

7 combinations appropriate for PCD as a ready-to-drink beverage, as 85°C for 20 seconds (60), as well as for a ready-to-dilute concentrate (°Brix > 44), as 75 °C for 25 minutes during a static process (64), have been reported.

Filling-Packaging. Following filtration, the pasteurized beverage is immediately filled in non- returnable empty PET (polyethylene terephthalate) or glass bottles and capped with metallic lug caps or Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 plastic closures. In this process, hot-fill technology is commonly used to prevent microbial contamination during the filling step, and to take advantage of the product while it is hot (around 85 °C) so as to reduce the microbial load from the inside of the primary packaging (caps and bottles) in combination with a 180° bottle inversion. In this manner, the packaging is correctly disinfected, and the beverage retains commercial sterility. Thereafter, the bottles are quickly cooled to avoid any alterations of the color or sensorial characteristics of the beverage. The cooling is achieved through the passage of units produced under a cooling tunnel, where the product normally exits at a temperature between 40 – 50 °C.

Subsequently, the water retained in different parts of the cap and the bottle is removed using filtered air cannons (air blowers). The cold product is now ready for its labeling, coded, stored and subsequent distribution.

FACTORS THAT IMPACT THE MICROBIOLOGICAL STABILITY OF THE BEVERAGE

Raw materials. Despite the fact that different formulas for preparing purple corn drink exist in

Peru, the following ingredients are commonly used in this beverage: potable water, purple corn, fruits

(apple, quince, and pineapple), spices (cinnamon and cloves), sugar or sweeteners, and acidity regulators

(lemon juice or organic acids). This traditional beverage may also include orange peel, pineapple peel, peach, anise and fig leaves, as well as the use of preservatives (potassium sorbate, sodium benzoate or a mixture of them) at the industrial level. The microbiological aspects of each raw material used in the production process that could affect the stability of the final beverage are detailed below:

At the industrial scale, potable water is a crucial component in the formulation of purple corn drink and must meet the standards recommended by the World Health Organization as well as local regulations in the area where the beverage is produced. Cryptosporidium parvum which has been linked to several illness outbreaks in drinking water (57) and fruit juices (63), should be given special attention.

Purple corn is the most essential ingredient in the formulation of the beverage because it Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 contributes to particular color (by anthocyanin content) and possesses high starch content. Corn is also a potential source of microbial contamination. As with all corns, the most common microorganisms are fungi (1), mainly Fusarium spp. (18), but spore-forming bacteria may be present as well. The most concerning aspect of fungi is that they can form mycotoxins (aflatoxins and fumonisins) as a byproduct of their metabolism (11), which are extremely difficult to remove during processing. For this reason, proper storage controls for purple corn must be implemented, with a focus on humidity and temperature conditions.

Fruits are the most significant source of microorganisms among all ingredients in beverage. Fruits like apple, quince and pineapple can contain high populations of microorganisms that can affect the stability of the final beverage (17, 62); lactic acid bacteria, acetic bacteria, molds, yeasts, and thermo- acidophilic endospore-forming bacteria being the most common spoilage agents (36, 51). Fruit juices and fruit-based non-carbonated beverages are prone to fermentation, sensory changes, phase changes caused by the presence of pectinolytic enzymes, turbidity, among others (25, 74), and the purple corn drink may be affected by these issues as well. Heat-resistant molds (Paecilomyces niveus, Paecilomyces variotti,

Aspergillus fischeri, among others) and Alicyclobacillus spp. (mainly Alicyclobacillus acidoterrestris) should be avoided or eliminated in the production of pasteurized purple corn drink. If present, they may survive the pasteurization process and cause undesirable changes during the storage of the final product

(54, 61). The main characteristics of these microorganisms is that their spores are extremely resistant to different thermal treatments, i.e., pasteurization (44, 49) and due to its acid tolerance, they can survival and growth vigorously at the low pH levels found in PCD (62). Given the inclusion of apples in the

9 beverage's formulation, special attention must be paid to Clostridium pasteurianum, an anaerobic, spore- forming bacteria capable of creating off-odors and off-flavors (due to the presence of butyric acid) as well as gas production (hydrogen, carbon dioxide) in acid products as part of its metabolism; thereby, resulting in swollen packages (19). C. pasteurianum spores have low probability of germination due to the intrinsic properties of final drinks (pH < 4.0); however, it could represent a potential problem just before the

standardization stage, where purple corn drink extract (whose pH value is typically greater than 5.0) is Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 stored and not yet acidified.

Spices (whole, in pieces, or powdered) are added to the beverage formulation during the boiling process to add a unique taste and aroma. Due their inherent properties, spices can indirectly enhance the functional and antimicrobial properties of this Peruvian beverage (13, 58). Spices contain a variety of phytochemicals, including phenolic compounds, flavonoids, isoflavonoids, coumarins, terpenes, and glycosides, which act as antioxidant and exhibit antimicrobial properties against harmful bacterial and fungal species (10, 13). The two spices that are usually incorporated in the formulation are cinnamon

(Cinnamomun verum) and clove (Syzygium aromaticum). Cinnamon quills, obtained from the central part of tree´s bark, is the product commonly commercialized (59) and contains essential oils such as monoterpenes, alcohols, monoterpene hydrocarbons, acids, aldehydes, phenols, and other compounds as lignas and sterols (9). While cloves contain several volatile oils, mainly eugenol, acetyl eugenol, β- caryophyllene, methyl salicylate, pinene and vanillin whose antibacterial and fungal properties have been demonstrated in several studies (20, 40). Various research have been carried out to demonstrate the antimicrobial properties of the active principles present in spices, however, these properties have not yet been validated in Chicha Morada. On the other hand, spices are regarded as a vehicle for a significant number of pathogenic and spoiling microorganisms due to their exposure to various environmental conditions during harvest, processing, and commercialization (14). Accordingly, verification of microbiological quality of spices in the factory (during the reception step) is critical before entering the production area.

Traditionally, sugarcane (Saccharum officinarum) has been incorporated into this beverage in order to add sweetness and body; however, in recent years, new formulations with sweetener replacement by sugar have been incorporated into the Peruvian beverage market to meet new consumer demands as well as to meet governmental health policy (8). Without a doubt, this replacement may have an impact on the microbiological stability of the final product due to a decrease in the content of solutes (sugar) and

increase of water activity. Accordingly, it should be evaluated in each formulation. At the industrial level, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 refined white sugarcane is more commonly used, although some formulations may use brown sugar. The production process of refined white sugar and its physico-chemical properties, primarily low water activity, allows for this ingredient to have a low microbial load, contributing to its microbiological stability during the storage and distribution (42). Therefore, refined white sugarcane does not pose a food safety risk (attributable to pathogenic microorganisms); however, it can be a potential source of spoilage microorganisms that can cause alterations in the final beverage (28). To reduce the risk of microbiological spoilage in finished products, the United States National Soft Drink Association established microbiological standards for industrial sugar, with a maximum of 200 CFU/10 g for mesophilic bacteria and a maximum of 10 CFU/10 g for molds and yeasts (69). Studies conducted in Brazil and Poland have confirmed that refined white cane sugar produced and commercialized in those countries during various campaigns meet microbial standards; thereby, highlighting the high quality of these products through the technological process applied (41, 69). Contrarily, an important group of bacteria capable of forming thermally resistant spores can be present in refined white cane sugar (26, 39), and can later cause potential problems in pasteurized purple corn drink during storage and distribution. The most common bacteria in refined white sugar are Geobacillus stearothermophilus and Bacillus coagulans which cause flat sour spoilage, Clostridium thermosaccharolyticum which can acidify canned foods and produce swelling, and

Desulfotomaculum nigrificans which cause “sulphur stinker” during the sulfide spoilage (4, 42, 56).

Sugar and various types of artificial sweeteners have also been replaced in food and beverages in recent years by natural sweeteners such as stevia, which have superior functional and sensory properties

(22, 70). Recent studies have shown that stevia glycosides present several advantages when added to food formulations, for example, steviosides improve the stability of potassium sorbate in a food system during storage and thus help to prevent the growth of two important osmotolerant and preservative-resistant yeasts (Zygosaccharomyces bailii and Z. rouxii) (27). However, more research into the antimicrobial effect of commercial stevia or their steviol glycosides against pathogenic bacteria, mainly in the same

foodstuffs is still necessary (5, 32). Another added benefit is the fact that stevia could stabilize the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 anthocyanins and other bioactive compounds presents in the purple corn drink, resulting in a synergistic effect during the shelf life (6). Studies on chokeberry juices have demonstrated improved levels of vitamin

C, total phenol content and the antioxidant capacity when compared to the same juices with sucrose added

(53).

Because of its nature and processing conditions; acidifying agents, such as citric acid and industrialized lemon juice; are not considered as potential sources of pathogenic bacteria. Instead, they perform as a hurdle lowering the pH of the beverage. However, a study considering lemon juice concentrate processing factors; such as soluble solids (6.20 to 68 °Brix), pasteurization temperature (82 to

95 °C) and pH (2.28 to 3.5); shows that Alicyclobacillus acidoterrestris can survive and remain viable in this ingredient (33). Therefore, Alicyclobacillus acidoterrestris should be controlled by processing industries.

Other ingredients (not commonly used in formulation) must be evaluated based on their nature, treatments used, and marketing strategy. Orange and pineapple peel, peach and fig leaves could all be classified as fruits, while anise is classified as a spice, each with its own unique microbial ecology.

Production stages. In general, each processing stage is a potential source of microbiological contamination, presumably due to poor hygiene practices in the factory, such as wrong time intervals between productions to carry out a new hygiene operation, inadequate washing and disinfection procedures, difficulty sanitizing the machine/equipment and annexes (pumps and pipes) with dead points

(where the turbulent flow is not achieved with the usual cleaning-in-place operation), among others. A

12 recent study on fruit juice processing factories from Europe and the United States revealed the need to establish new sanitation procedures based on more rigorous standards, with an emphasis on preventing contamination by spoilage microorganisms (55). For example, fermentation episodes caused by

Saccharomyces cerevisiae yeast have been reported in this beverage (67), and their presence has been linked to problems with closure or probable post-pasteurization contamination. In this specific spoilage

incident, units of swollen packages were detected in the factory warehouse days after its production, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 showing abundant gas production, deformation of plastic bottle, high internal pressure, increase in headspace, presence of sediment and a characteristic off-odor and off-flavor not typical of the original drink, while the color was apparently unaffected. It is known that these spoilage incidents occur in PCD, but unfortunately, it is not very documented.

Various microbiological problems linked to fermentation and the presence of visible molds in non-carbonated beverages are caused by aciduric bacteria, and mold and yeasts, which enter the product through the filling valves or the capper (after heat treatment) (31). For this reason, a generalized control measure in beverage factories is the microbiological evaluation of the rinse water coming from filler valves. In order to prevent contamination and taking into account the recommendation made by Lawlor and Leighton (30), a special microbiological sampling program that focuses on the rinse waters of filler valves (post cleaning-in-place) should be implemented in the factory of this Peruvian beverage with maximum values of 15 CFU aciduric bacteria, mold or yeast/100 ml.

The air and food handlers are two other relevant environmental factors to consider during the production of purple corn drink (15, 16) to avoid a cross-contamination. A filling room sub-area should be established within the production line, where air quality is improved through filters with high particulate retention efficiency. Food handlers, on the other hand, must follow a rigorous food safety training program, highlighting mainly the correct way to intervene the equipment due to stops or problems related to maintenance during the production.

Taking into account the process production designed for this beverage, a particular and specific control must be carried out on four points. First, because purple corn drink extract is highly susceptible to microbiological contamination due to its high pH value (typically > 5.0), it should not be stored for an extended period of time before the next process stage. Second, the storage of purple corn drink (before pasteurization), due to long holding times, could increase the initial microbiological load of this product

with subsequent sensory changes, given that the beverage already contains sugar and other nutrients for Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 the development of mold and yeasts at this stage. Third, the fundamental step of the capping process that maintains the product stable throughout its shelf-life, must have specific controls and adequate limits for pull-up, torque, and vacuum and pressure testing, ensuring a proper seal and the absence of product leaks.

Finally, a fourth point is linked to microbiology of cooling water, because at this stage, small droplets of water may enter the product through micro spaces between the bottle and cap that are still finishing sealing to create the corresponding vacuum. Chemical agents against microorganisms and algae must be added to this water to maintain a low load of heterotrophic bacteria (below 500 CFU/ml), molds, yeasts, and the absence of total coliforms. A recommendation for factories is the control of total hardness in the water used in the cooling tunnel because high hardness values can interfere with the functionality of chemical agents, and, on the other hand, form layers of inorganic salts in different parts of the tunnel

(primarily in the nozzles), contributing to formation of biofilms.

Forms of commercialization. Ready-to-drink Purple corn drink is typically sold in two types of packaging: glass and PET bottles, both of which establishes a form of preservation through a physical barrier against oxygen and ensures a specific shelf-life (30). These products are held at room temperature under shade during distribution; however, this can change during its sale in markets, supermarkets and other types of channels, such as street vendors. When sold in supermarket and grocery stores, this beverage is commonly stored under refrigeration, especially during the summer season. Foodborne pathogens are able to adapt and tolerate high acid environments causing diseases outbreaks if other hurdles are not properly placed in the formulation, in the process or in the product packaging (7, 38, 68).

A study carried out by Ramos Guerrero et al. (47) investigated the survival of pathogenic bacteria in purple corn drink (°Brix: 12.2, pH: 3.29) stored at 8 + 1 °C and found that Salmonella enterica was reduced in 1.31 log during the first 48 h of storage, not being detectable (<1 CFU/ml) after 120 h. Its intrinsic properties, such as low pH, presence of anthocyanins, organic acids and phytochemical compounds from fruits and spices, serve as synergistic hurdles against these types of microorganisms;

however, refrigeration temperatures could represent a potential problem for the safety of this product, due Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 to increases survival times of foodborne pathogens in this type of beverages (7, 24).

The low pH of the purple corn drink is a limiting factor for many foodborne bacteria, however, allows survival and growth of important groups of aciduric microorganisms. This type of microorganisms can alter the product causing undesirable physical-chemical and sensory changes in the drink. Yeast, molds, lactic acid bacteria and acetic bacteria can consume the nutrients present in the beverage during storage or distribution changing the properties of the final product, as it happens in drink containing fruit juices. Unfortunately, there is not much information available about the microbial ecology in PCD. For this, specific studies must be carried out in order to elucidate the behavior of these microorganisms on

PCD and observe if its metabolism can be altered when the type of organic acid change in the formulation or the anthocyanin level is modified.

As with other non-carbonated beverages, various microbiological issues can be associated with the distribution and sale of this type of beverage, with the main focus being on how the product is stacked and its exposure to unfavorable conditions during its storage (31). It is important to highlight that, the packaging must be secured at all times in order to preserve the products’ airtightness. The way the products are stacked is crucial; many cases of microbiological contamination can be traced back to this step. Over stacking and improper handling during distribution (such as knocking or banging) cause a weakening in the structure of package, resulting in deformations, cracks, crushing, and other problems, as well as product contamination. Metallic caps on glass bottles are more susceptible to mechanical damage than plastic closures on PET bottles. The final contamination of the beverage is caused by a pressure

15 difference between the product and the surrounding environment. The type and load of microorganisms present in this environment, as well as the type of leak in the packaging, will determine the form of spoilage.

CONCLUDING REMARKS

Chicha Morada is a Peruvian specialty beverage with a particular manufacturing process. It stands Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 out from other non-carbonated beverages by its attractive purple color, distinctive flavor and smell. Due to the nature of its raw materials and the design of its production, this beverage has more advantages than disadvantages in terms of microbiological stability. Because it is an acidified drink and the conditions of its production process, its microbial ecology is primarily limited to molds, yeasts and aciduric bacteria, but it is vitally important to avoid the entry of heat-resistant molds and Alicyclobacillus spp. Extra caution must be taken to prevent microbiological spoilage caused by cross-contamination with equipment, food handlers or other environmental factors. Furthermore, microbiological monitoring of the water used in the cooling tunnel must be performed on regular basis, keeping in mind that it has a high likely of contaminating the beverage (post-pasteurization). Another point worth highlighting is the precautions needed during the capping process and distribution to avoid damaging or causing irregularities on the primary packaging (bottle and cap). All of these preventative controls, will help to improve the HACCP system of pasteurized purple corn drink in the factories dedicated to its production, ensuring its quality and safety.

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FIGURE LEGENDS

FIGURE 1. Flowchart of Peruvian purple corn drink (“Chicha Morada”) at industrial scale.

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TABLE 1. Nutritional values of Peruvian purple corn and purple corn drink.

Values expressed by 100 g of sample Parameter Purple corn Purple corn drink

(without corncob) (with sugar) Energy (kcal) 355 20 Water (g) 11.4 95 Proteins (g) 7.3 0.0 Total fat (g) 3.4 0.0

Total carbohydrates (g) 76.2 4.9 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 Ash (g) 1.7 0.1 Calcium (mg) 12 24 Phosphorus (mg) 328 4 Iron (mg) 0.20 1.3 Thiamin (mg) 0.38 - Riboflavin (mg) 0.22 0.1 Niacin (mg) 2.84 0.04 Vitamin C (mg) 2.10 1.90 (-) Unreported value.

All nutritional values were extracted from Tablas Peruanas de Composición de Alimentos, 10th edition

(48).

Revised Figure 1 Click here to access/download;Figure;Fig. 1.docx

FIGURE 1 A. Preparation of Receipt of ingredients Receipt of raw purple corn (spices, sugar, Receipt of materials drink extract sweeteners, acidity packaging (purple corn regulators and materials and fruits) preservatives)

Storage Storage Storage

Downloaded from http://meridian.allenpress.com/jfp/article-pdf/doi/10.4315/JFP-21-190/2879730/jfp-21-190.pdf by guest on 28 September 2021 Fractionation Fractionation

Washing and brushing

Cutting-up Spices Boiling Potable water

Filtered 1

B. Standardization Blending and homogenization

C. Thermal Pasteurization inactivation

D. Filling- Filtered 2 Packaging

Hot filling

Capping

Product cooling

Labeling and packaging

Final storage

Tracked Changes

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Click here to access/download Tracked Changes Blank 1.docx Response to Reviewers

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Click here to access/download Response to Reviewers Blank 2.docx Copyright Release

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