Individual Project FSCN 4312 Fall 2014 Name Fruit‐Flavored Yogurt

Individual Project

FSCN 4312

Fall 2014

Name

Fruit‐Flavored Yogurt

II. Summary

Fruit‐flavored yogurt is a fermented milk product cultured with lactic‐acid producing bacteria, including Lactobacillus bulgaricus and Streptococcus thermophiles, and flavored with the addition of whole fruits, fruit purees, or fruit variegates. Optional ingredients may include vitamins, stabilizers, and colorants, among others. The percent milkfat of the yogurt prior to addition of fruits and flavors must be a minimum of 3.25%. Percent milk solids not fat must be a minimum of 8.25%, and titratable acidity (percent lactic acid) must be a minimum of 0.9%.

Raw ingredients one may find in fruit‐flavored yogurt include cream, milk, lowfat milk, or skim milk (or a combination of these), lactic‐acid producing bacteria (Lactobacillus bulgaricus and Streptococcus thermophiles), sugar or other sweeteners, stabilizers, fruit and fruit flavorings, color additives, and vitamins. In the example used for the analysis at present, the four ingredients in greatest abundance were cultured, pasteurized grade A lowfat milk, sugar, strawberries, and modified corn starch. The following components of pasteurized grade A milk are tested: milkfat, milk solids not fat, protein, and lactose (all by mid infrared spectroscopy), total plate count (by Standard Plate Count Method), somatic cell count (by Direct Microscopic Somatic Cell Count, DMSCC), coliform count (by Petrifilm Coliform Count Method), beta lactam antiobiotic residues (by Quantitative Bacillus stearothermophilus Disc Method), temperature, titratable acidity (by phenophthalein indicator), and pasteurization (Scharer Spectrophotometric ALP Method). Tests performed on strawberries (typically in the form of a strawberry variegate), sugar, and modified corn starch are likely done by the supplier as a measure to meet the requirements of the certificate of analysis (COA), but could also be performed by the yogurt manufacturer. Sugar would be tested for moisture, polarization, invert sugar content, sulfated ash, and color. Strawberry variegate would typically be tested for Brix, titratable acidity, viscosity, total aerobic plate count, yeast and mold count, and coliform count in order to meet manufacturer specifications. Finally, corn starch may be tested for pH, sulfur dioxide, residual solvents, oxidants, percent ash, percent moisture and, as a complementary measure, iron; these tests would be accompanied by additional microbiological tests.

During processing, samples are pulled out at three different stages for quality testing. After the first step, wherein all ingredients save fruits, fruits flavorings, and cultures, are blended, samples are pulled for tests on milkfat and total solids not fat by mid infrared radiation analysis. The succeeding steps include pasteurization of the milk blend, homogenization, cooling, and inoculation with cultures. The remaining order of processing depends on whether Swiss style yogurt or set style yogurt is being produced. If Swiss style yogurt is desired, fermentation precedes the addition of fruits and fruit flavorings; if set style yogurt is desired, unfermented yogurt is poured over a layer of fruit and then fermentation occurs. In either case, samples are pulled out after fermentation for tests on pH or titratable acidity (titratable acidity is tested by the phenophthalein indicator method) and after addition of fruits and fruit flavorings for tests on Brix (by the Spindle method). Both processing schemes end with blast cooling and refrigerated storage and distribution.

Samples of the final fruit‐flavored yogurt product are withdrawn for testing of several components. Milkfat of yogurt may be tested by the Mojonnier method, total solids not fat may be determined by the difference in total fat content and total solids content (by forced draft oven), and protein may be determined by the Kjeldahl method. Microbiological testing includes a coliform count by Petrifilm High‐ Sensitivity Coliform Count Plates and a yeast and mold count by the Hydrophobic Grid Membrane Filter Method. Viscosity, titratable acidity (percent lactic acid), and calcium where desired are additionally measured. Percent lactic acid can be determined by a spectrophotometric method, while calcium is often determined by Atomic Absorption Spectrophotometry.

III. Summary of Standard of Identity

Yogurt is the product of culturing cream, milk, partially skimmed milk, skim milk, or a combination of these products with a culture containing Lactobacillus bulgaricus and Streptococcus thermophiles, both of which produce lactic acid. Optional ingredients that may be added at the time of culturing include vitamins (e.g. vitamin A or D), ingredients intended to decrease the total nonfat solid content (e.g. concentrated skim milk, nonfat dry milk, buttermilk, whey, lactose, lactalbumins, lactoglobulins or modified whey), most nutritive carbohydrate sweeteners (with the exception of blackstrap molasses and table syrup), flavor additives, color ingredients, and stabilizers. Prior to any flavors (including fruit flavors) being added to the yogurt, percent milkfat must be at least 3.25, percent milk solids not fat content must be at least 8.25, and titratable acidity (lactic acid) must be at least 0.9 percent. Homogenization and pasteurization or ultrapasteurization of the product is required before the addition of bacterial culture and may be conducted on the final product to control microbial growth (21 CFR 131.200).

IV. Nutritional Composition

Nutrient Composition of Fruit‐Flavored Yogurt

Declared Actual (rounded) Actual % (rounded) Amount Values of Daily Value % Daily Per Amount Per Daily Per Value Per Nutrient Servinga,b Servinga,c Valuec Servinga,c Servinga,c

Calories 168 Cal 170 Cal Calories from fat 17.6 g 20 Cal Total fat 1.95 g 2.0 g <65 g 3.00% 3% Saturated fat 1.261 g 1.0 g <20 g 6.31% 5% Cholesterol 8 mg 10 mg <300 mg 2.67% 3% Sodium 90 mg 90 mg <2400 mg 3.75% 4% Total carbohydrate 31.69 g 32 g 300 g 10.56% 11% Dietary fiber 0.0 g 0 g 25 g 0.00% 0% Sugars 31.69 g 32 g Protein 6.77 g 7.0 g 50 g 135.40% 14% Vitamin A 68 IU ‐‐ 5,000 IU 1.36% 2%

Vitamin C 1.0 mg ‐‐ 60 mg 1.67% 2% Calcium 235 mg ‐‐ 1000 mg 23.50% 25% Iron 0.10 mg ‐‐ 18 mg 0.56% 0% aServing size: 6 oz. (178g) bNutritional data obtained from USDA National Nutrient Database for Standard Reference, Release 27, 2014 cDaily Values and rounding rules obtained from: 21 CFR 101.9

V. Product Description

Fruit‐flavored yogurt is a creamy and viscous fermented milk product with fruit and/or fruit flavorings either blended in (Swiss style yogurt) or on the bottom (set style yogurt).

VI. Raw Ingredients

A. List of raw ingredients (Yoplait Original: Strawberry Banana) Cultured Pasteurized Grade A Lowfat Milk, Sugar, Strawberries, Modified Corn Starch, Nonfat Milk, Banana Puree, Kosher Gelatin, Citric Acid, Natural Flavor, Tricalcium Phosphate, Pectin, Colored with Carmine, Vitamin A Acetate, Vitamin D3. With active yogurt cultures including L. Acidophilus.

B. Tests on raw ingredients: Sampling: randomly selected packages of milk product are collected and placed in a sample case with a temperature control sample at 0‐4.4°C; analysis should being before 48 hours. If milk is not packaged in individual containers, 100mL of thoroughly mixed product is transferred to a sample case for analysis (Hooi et al., 2004). Tests on sugar, strawberries, and modified corn starch are usually performed by the supplier to comply with their certificate of analysis, but may also be performed by the yogurt manufacturer, in which case representative samples of each are pulled for analysis. 1. Cultured, Pasteurized Grade A Lowfat Milk: Milkfat Content a. Test: Mid Infrared Spectroscopy (MIR) b. Reference: Hooi et al., 2004; Schoenfuss (Personal Communication); AOAC, 2012, Method 972.16 c. Reason for Measuring: The milkfat content of milk must be measured in order to comply with the final standard of identity of yogurt, as well as manufacturer specifications. d. Principle: The infrared instrument first homogenizes the sample, then bombards it with infrared radiation at a wavelength (or range of wavelengths) corresponding to the wavelength at which the component of interest (e.g. fat) absorbs light. Radiation of a reference wavelength just outside the optimal range of the functional group (where only a small amount of light is absorbed by the component) is also used. To determine fat content, wavelengths are chosen based on the frequency at which the carbonyl groups of ester bonds in glycerides and/or the methyl groups of fatty acids absorb light. Carbonyl groups absorb light between 5.72 and 5.76µm (reference wavelength of 5.58µm), and methyl groups absorb light between 3.48 and 3.51µm (reference wavelength of 3.57µm). Absorption can be correlated to concentration of the component using a standard established by a primary method. 2. Cultured, Pasteurized Grade A Lowfat Milk: Milk Solids Not Fat a. Test: MIR b. Reference: Hooi et al., 2004; Schoenfuss (Personal Communication); AOAC, 2012, Method 972.16 c. Reason for Measuring: Milk solids not fat must be measured in order to comply with the standard of identity for the final product, yogurt, and to meet manufacturer specifications. d. Principle: See VI.B.1.d. Once primary values of individual components have been calculated, these values can be used to determine total solids not fat in the sample. 3. Cultured, Pasteurized Grade A Lowfat Milk: Protein a. Test: MIR b. Reference: Hooi et al., 2004; Schoenfuss (Personal Communication); AOAC, 2012, Method 972.16 c. Reason for Measuring: Protein may be measured to meet manufacturer specifications. d. Principle: See VI.B.1.d. Secondary amide groups absorb optimally between 6.46 and 6.60µm; a reference wavelength of 6.7µm may be used. 4. Cultured, Pasteurized Grade A Lowfat Milk: Lactose a. Test: MIR b. Reference: Hooi et al., 2004; Schoenfuss (Personal Communication); AOAC, 2012, Method 972.16 c. Reason for Measuring: Lactose is the primary carbohydrate/sweetener in milk. It is also important in final determination of total solids not fat. d. Principle: SeeVI.B.1.d. To determine lactose content, wavelengths are chosen based on the frequency at which the hydroxyl groups of lactose absorb light, which falls in the range of 9.47 and 9.61µm; a reference wavelength of 7.7µm may be used. 5. Cultured, Pasteurized Grade A Lowfat Milk: Total Plate Count a. Test: Standard Plate Count (SPC) Class O or Petrifilm Aerobic Count (PAC) Class A1 b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Hooi et al., 2004. c. Reason for Measuring: Total plate count is measured to determine quality of a milk product, as well as to estimate microbial presence and test for contamination. d. Principle: In the SPC method, measured milk samples are applied to standard methods agar (SMA) and incubated for 48 ±3 hours at 32±1°C in the presence of oxygen. Milk samples may be applied directly or diluted first. After incubation, plates with 25‐250cfu/mL are used to determine number of microorganisms per mL/gm of sample. The PAC method does not present data of a statistically significant difference compared to SPC data. In the PAC method, 1mL of sample is pipetted onto a culture medium consisting of two plastic films coated with adhesive, powdered nutrients, dehydrated cold water‐soluble gelling agent, and an indicator dye. The sample is applied below the first film, which then covers the sample to evenly distribute it across the plate. The plates are incubated under the same parameters as SPC. 6. Cultured, Pasteurized Grade A Lowfat Milk: Somatic Cell Count a. Test: Direct Microscopic Somatic Cell Count (DMSCC) or Electronic Somatic Cell Count (ESCC) b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Hooi et al., 2004. c. Reason for Measuring: Somatic cell count is used to test for abnormal milk; the presence of somatic cells (or leucocytes) is indicative of mastitis or other conditions that result in inflammation of udder. d. Principle: A homogenized and representative milk sample is applied to a microscopic slide, and typical microscopic techniques are used to quantitatively measure the number of somatic cells per mL of milk. 7. Cultured, Pasteurized Grade A Lowfat Milk: Coliform Count a. Test: Coliform Plate Count, Petrifilm Coliform Count (PCC), or High Sensitivity Coliform Count (HSCC) b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Hooi et al., 2004; AOAC, 2012, Method 986.33. c. Reason for Measuring: The coliform count is used to test for post‐pasteurization contamination by coliform organisms. d. Principle: 1mL of sample is applied to a 20cm2 area on a PCC plate consisting of 2 plastic films coated in violet red bile nutrients, a dehydrated cold water‐ soluble gelling agent, and tetrazolium indicator (a pH indicator). The plate is incubated at 30‐37°C to measure total coliforms and 41‐45°C to measure fecal coliforms. Coliforms produce acid on the PCC plate, which causes the gel to change color, enabling quantitation of cell count. 8. Cultured, Pasteurized Grade A Lowfat Milk: Beta Lactam Antibiotics in Milk a. Test: Quantitative Bacillus stearothermophilus Disc Method b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Standard Hooi et al., 2004; AOAC, 2012, Method 982.16 c. Reason for Measuring: The Quantitative Bacillus stearothermophilus Disc Method tests for any beta lactam antiobiotic residues in milk. If a sample tests positive, the milk must be rejected per regulatory requirements. d. Principle: A paper disc saturated with milk is introduced to a medium inoculated with B. stearothermophilus; the plate is incubated at 64°C, which encourages the growth of B. stearothermophilus. Where no inhibitors are present, the bacterial spores will produce acid that cause the indicator in the medium to change from purple to yellow. Where inhibitors (antibiotics) are present, the medium does not change color; the zone of inhibition must be greater than 16mm for the test to be considered positive. 9. Cultured, Pasteurized Grade A Lowfat Milk: Temperature a. Test: Temperature following cooling post‐pasteurization should not exceed 45°C. When raw milk is originally received, it should not exceed 10°C. b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Tamime and Robinson, 1999. c. Reason for Measuring: The temperature of milk should be measured per regulatory requirements. 10. Cultured, Pasteurized Grade A Lowfat Milk: Titratable Acidity a. Test: Phenolphthalein Indicator (Class O) b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Hooi et al., 2004 c. Reason for Measuring: Titratable acidity is measured to comply with the standard of identity for milk and the final product, as well as to meet manufacturer specifications. d. Principle: A known amount of sample is added to an Erlenmeyer flask along with phenolphthalein indicator and titrated with a standardized solution of 0.1N sodium hydroxide until the sample solution turns a light pink. The total volume of titrant used can be used to calculate total lactic acid in the sample. 11. Cultured, Pasteurized Grade A Lowfat Milk: Pasteurization a. Test: Scharer Spectrophotometric ALP (alkaline phosphatase) Method b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Hooi et al., 2004; AOAC, 2012, Method 946.01 c. Reason for Measuring: The phosphatase test is conducted to test for efficiency of pasteurization. d. Principle: Alkaline phosphatase (ALP) is a thermodynamically stable enzyme; pasteurization is effective once this enzyme has been inactivated. ALP activity yields phenol compounds from the substrate, phenyl phosphate.The activity of ALP in milk can be determined by reacting any phenol product with Gibbs reagent, which forms a CQC‐phenol complex that can be measured spectrophotmetrically; these measurements are compared to those obtained from phenol standards that have been complexed with the Gibbs reagent. 12. Sugar: Moisture a. Test: Vacuum Drying b. Reference: AOAC, 2012, Method 925.45; Malayan Sugar Manufacturing, 2008 c. Reason for Measuring: Moisture of sugar is determined to meet manufacturing specifications as part of the COA. d. Principle: 2‐5g of sample is weighed in a flat dish and dried in a vacuum oven for 2 hours at ≤70°C and ≤50mmg Hg. After drying, it is cooled in a dessicator, and the final weight of product is recorded. Difference in weight before and after drying is used to determine percent moisture. 13. Sugar: Polarization a. Test: Immediate Direct Polarization b. Reference: AOAC, 2012, Method 920.182; Malayan Sugar Manufacturing, 2008 c. Reason for Measuring: Polarization of sugar is determined to meet manufacturing specifications as part of the COA. d. Principle: Polarization of sugar is determined using a polarimeter. Sample is added to a 100mL volumetric flask with 5mL of alumina cream and diluted to volume with water at 20°C; sample is then filtered into a 200mm tube in order to be analyzed by the polarimeter. 14. Sugar: Invert Sugar a. Test: Lane‐Eynon General Volumetric Method b. Reference: AOAC, 2012, Method 923.09; Malayan Sugar Manufacturing, 2008 c. Reason for Measuring: Invert sugar is determined to meet manufacturing specifications as part of the COA. d. Principle: percentage of invert sugar is calculated by determining how much of a sugar solution is needed to reduce all Cu ions in the Soxhlet mixture, consisting of hydrated copper sulfate and hydrated alkaline tartrate. The methylene blue indicator will be coloreless when all Cu ions have been reduced, indicating the end of the titration; the solution will be a light orange. 15. Sugar: Sulfated Ash a. Test: Ash of Sugars and Syrups, Sulfated Ash b. Reference: AOAC, 2012,Method 900.02 Method G; Malayan Sugar Manufacturing, 2008 c. Reason for Measuring: Sulfated ash of sugar is determined to meet manufacturing specifications as part of the COA; amount of sulfated ash must fall below acceptable maximum. d. Principle: A mixture of 5g sample and 5mL (10% by weight) sulfuric acid are mixed, heated on a hot plate to carbonize, and then ashed in a muffle furnace at 550°C. The percentage of ash remaining is representative of percent sulfated ash. 16. Sugar: Color a. Test: Spectrophotometric Method b. Reference: AOAC, 2012, Method 954.10; Malayan Sugar Manufacturing, 2008 c. Reason for Measuring: The color of sugar is determined to meet manufacturing specifications as part of the COA. d. Principle: Color is determined spectrophotometrically by the wavelength or range of wavelengths at which the sugar solution best absorbs. 17. Strawberries (Aseptic Strawberry Variegate): Brix a. Test: Spindle Method b. Reference: Schoenfuss (Personal Communication); AACCI Method 80‐53.01; TreeTop Fruit Ingredients, 2014; Oregon Fruit Products Co., 2010 c. Reason for Measuring: Brix of the aseptic strawberry variegate is measured to meet manufacturer specifications as part of the COA. d. Principle: A Brix, or Baume hydrometer can be used to find the density of fruit juices, molasses, and syrups by measuring the solids from sugar. The Brix scale divulges the percentage of weight of pure sucrose in pure solutions. 18. Strawberries (Aseptic Strawberry Variegate): Titratable Acidity a. Test: Indicator Method, Glass Electrode Method b. Reference: AOAC, 2012, Method 942.15; Schoenfuss (Personal Communication); TreeTop Fruit Ingredients, 2014; Oregon Fruit Products Co., 2010 c. Reason for Measuring: Titratable acidity of the aseptic strawberry variegate is measured to meet manufacturer specifications as part of the COA. d. Principle: With the indicator method, extra dilution for highly colored solutions allows the color change to be visualized. The solution is diluted as appropriate with CO2 free water and titrated with 0.1N alkali solution. For every 100mL of diluted sample, 0.3mL of indicator is used. Percent acid can be calculated using the factor specific to the acid of greatest concentration in the fruit product. The glass electrode method may also be used. Essentially, a glass electrode measures the pH of the solution after a known amount of 0.1M alkali titrant is added in equal intervals. After pH of 7 is reached, only four drops of titrant should be added at a time; this can be increased to greater than four drops after a pH of 8.1 is reached. 19. Strawberries (Aseptic Strawberry Variegate): Viscosity a. Test: Viscosity (Apparent Consistency) of Fruit Products b. Reference: AOAC, 2012, Method 967.16; TreeTop Fruit Ingredients, 2014; Oregon Fruit Products Co., 2010 c. Reason for Measuring: Viscosity is measured to meet manufacturer specifications as part of the COA. d. Principle: Sample is run through a capillary viscometer, and the time for the meniscus of the sample to reach the calibration line of the capillary tube is recorded. 20. Strawberries (Aseptic Strawberry Variegate): Total Aerobic Plate Count a. Test: Hydrophobic Grid Membrane Filter b. Reference: AOAC, 2012, Method 986.32; TreeTop Fruit Ingredients, 2014; Oregon Fruit Products Co., 2010 c. Reason for Measuring: Total aerobic plate count is measured to meet manufacturing specifications and test for bacterial contamination. d. Principle: Hydrophobic residues prevent the spread of bacterial colonies on a membrane filter. The membrane filter is applied to a plate, to which the sample is introduced, and the plate is incubated. Squares positive for colonies are enumerated and the number of positive squares is converted to most probable number (MPN) of microorganisms. 21. Strawberries (Aseptic Strawberry Variegate): Yeast and Mold Count a. Test: Hydrophobic Grid Membrane Filter (ISO‐GRID) Method Using YM‐11 Agar b. Reference: AOAC, 2012, Method 995.21; TreeTop Fruit Ingredients, 2014; Oregon Fruit Products Co., 2010 c. Reason for Measuring: Yeast and mold counts are measured to meet manufacturer specifications as part of the COA. d. Principle: The sample suspension is administered over a membrane filter that has been treated with hydrophobic residues that prevent the spread of colonies across gridlines. Filters are introduced to a YM‐11 plate and incubated for 50±2 hours at 25±1°C. Squares positive for colonies are enumerated and the number of positive squares is converted to most probable number (MPN) of microorganisms. 22. Strawberries (Aseptic Strawberry Variegate): Coliform Count a. Test: Rapid Enumeration of Coliforms in Foods b. Reference: AOAC, 2012, Method 2000.15; TreeTop Fruit Ingredients, 2014; Oregon Fruit Products Co., 2010 c. Reason for Measuring: Coliform count of the aseptic strawberry variegate is measured to meet manufacturer specifications as part of the COA. d. Principle: Suspension is prepared by homogenizing 50g of sample with 450mL of water in a blender and bringing the suspension to a pH of 6.5‐7.5 with 1M NaOH solution. The suspension is added with a plastic spreader to a dry Petrifilm RCC plate over 20cm2 area and after the gelling agent has solidified, the plates are incubated for 24 hours at 35±1°C. Coliforms produce acid on the PCC plate, which causes the gel to change color, enabling quantitation of cell count. 23. Modified Corn Starch: Hydrogen‐Ion Activity (pH)‐‐Electrometric Method a. Test: Rapid Enumeration of Coliforms in Foods b. Reference: Farmal, 2007; AACCI Method 02‐52.01 c. Reason for Measuring: pH of modified corn starch is determined to meet manufacturer specifications set in the COA. d. Principle: Once the pH meter has been calibrated, the electrode is inserted into a thoroughly blended sample, and the pH is read by the meter. 24. Modified Corn Starch: Sulfur Dioxide (Residual Solvents) a. Test: Monier‐Williams b. Reference: Farmal, 2007; Corn Refiners Association, 2014. c. Reason for Measuring: Sulfur dioxide is measured to meet manufacturer specifications as part of the COA. Sulfur dioxide and other residual solvents can have detrimental health effects on the consumer. d. Principle: Sulfur dioxide gas is collected after a sample dispersed in an acidic solution is boiled. The sulfur dioxide gas is then oxidized to sulfuric acid in the presence of hydrogen peroxide. The sulfuric acid is subsequently titrated with a standardized alkaline solution, and the amount of titrant can be used to determine the original amount of sulfur dioxide in the sample. Wet‐milling can introduce several solvents that may reside after processing of the corn starch product. Regulated residuals in modified starches include arsenic, chloride, crude fat, lead, protein, sulfur dioxide, toluene, trichloroethylene, propylene chlorohydrin, manganese, acetyl groups, and phosphate. 25. Modified Corn Starch: Percent Ash a. Test: Ash in Starch b. Reference: Farmal, 2007; AACCI Method 08‐17.01 c. Reason for Measuring: commercial corn starches are known to contain a miniscule, but significant, amount of inorganic salts which may affect the overall nutrition (labeling) of the final product; mineral content may also affect the functionality of other components in the food matrix. d. Principle: Total ash is determined by ignition of the modified corn starch at high temperatures to combust all organic matter. The final weight as a percentage of the initial weight is representative of percent ash. 26. Modified Corn Starch: Percent Moisture a. Test: Modified Vacuum Oven Method b. Reference: Farmal, 2007; AOAC, 2012, Method 925.09; AACCI Method 44‐40.01; Bradley, 2010 c. Reason for Measuring: Knowing the percent moisture of the corn starch, as well as its activity of water (aw) is pivotal to estimating its shelf life, as well as the shelf life of the final product. The percent moisture is also determined to meet manufacturing specifications as part of the COA. d. Principle: The boiling point of water is decreased when pressures in the vacuum oven are lowered to ≤25mm Hg; this prevents carmelization of sugars in the sample and release of bound water. The sample is heated in the vacuum oven to 98‐100°C for approximately 5 hours, and the final and initial weights of the sample are used to calculate percent moisture.

VII. Processing of Product

A. Schematic Processing Diagram for Fruit‐Flavored Yogurt

All ingredients excluding fruits, flavorings, and cultures, blended together. Ingredients that may be added at this time include vitamins, ingredients intended to decrease total nonfat solids (e.g. whey), nutritive carbohydrate sweeteners, and stabilizers.

Pull out samples Ingredients added: Lowfat milk, sugar, for analysis of modified corn starch, nonfat milk, kosher milkfat (VII.B.1) gelatin, citric acid, tricalcium phosphate, and total solids pectin, carmine, vitamin A acetate, vitamin D3 not fat (VII.B.2)

Pasteurization of milk at 185°F (85°C) for 30 Pasteurization of milk at 185°F (85°C) for min. or 203°F (95°C) for 10 min. 30 min. or 203°F (95°C) for 10 min.

Homogenization of milk at 2000‐2500 psi, 60°C to thoroughly blend all ingredients.

Cooling: Yogurt is cooled to 4°C. If the yogurt is placed in storage after cooling, it may be subsequently heated to 45°C to create environmental conditions suitable to desired growth of cultures.

SET STYLE YOGURT STIRRED STYLE YOGURT

(FRUIT ON THE BOTTOM) (SWISS STYLE)

Batch or in‐line inoculation: Cooled yogurt mixture is Ingredients Inoculation: Cooled yogurt mixture is inoculated with inoculated with starter cultures, which include lactic acid‐ added: active starter cultures, which include lactic acid‐producing producing bacteria. Cultures may include Lactobacillus yogurt cultures, bacteria. Cultures may include Lactobacillus bulgaricus, bulgaricus, Streptococcus the rmophiles, Lactobacillus including L. Streptococcus thermophiles, Lactobacillus acidophilus, acidophilus, etc. or a combina tion of bacterial cultures. Acidophilus etc. or a combination of bacterial cultures.

Fermentation: inoculate yogurt is fermented Addition of fruit and flavorings. Yogurt is layered over fruit in packaging container. in a tank at 108°F (42°C) until pH of 4.5 is achieved (or desired titratable acidity).

Pull out Ingredients added: strawberry samples for Pull out variegate, banana puree samples for analysis of analysis of Brix (VII.B.4) pH/TA(VII.B.3)

Fermentation: inoculat e yogurt is fermented Addition of fruit and flavorings. Swiss style yogurt in a tank at 108°F (42°C) until pH of 4.5 is achieved by mixing fermented yogurt with the fruit achieved (or desired titratable acidity). blend. Pull out Pull out samples for samples for Ingredients added: strawberry analysis of analysis of variegate, banana puree Brix (VII.B.4) pH/TA(VII.B.3

Blast cooling to 4°C Blast cooling to 15°C

Refrigerated storage and distribution. Refrigerated storage and distribution.

Figure 1. Schematic Processing Diagram for Fruit‐Flavored Yogurt. References: Schoenfuss (Personal Communication); Chandan and Shahani, 1993; The Milk Quality Improvement Program; Hoolasi, 2005.

B. Tests During Processing: Sampling: Samples are pulled for testing of milkfat content and total solids not fat after the first step in processing, when which milk, stabilizers, vitamins, colorants, etc. are blended. Samples are also pulled for analysis after fermentation, during which time pH or titratable acidity are measured. Finally, samples are analyzed for Brix after the addition of fruits and fruit flavorings (Schoenfuss, personal communication; Chandan and Shahani, 1993). Samples intended for microbiological analysis are aseptically obtained, maintained at a temperature of 1‐5°C, and analyzed within three hours. Chemical analyses are performed immediately after pulling samples (Hoolasi, 2005). 1. Milkfat Content a. Test: MIR b. Reference: Hooi et al., 2004; Schoenfuss (Personal Communication); AOAC, 2012, Method 972.16 c. Reason for Measuring: The milkfat content is tested during processing to ensure the product will comply with the standard of identity for yogurt, as well as manufacturer specifications. d. Principle: See VI.B.1.d. 2. Total Solids Not Fat a. Test: MIR b. Reference: Hooi et al., 2004; Schoenfuss (Personal Communication); AOAC, 2012, Method 972.16 c. Reason for Measuring: The total solids not fat content is tested during processing to ensure the product will comply with the standard of identity for yogurt, as well as manufacturer specifications. d. Principle: See VI.B.2.d. 3. Titratable Acidity a. Test: Phenolphthalein Indicator (Class O) b. Reference: Grade A Pasteurized Milk Ordinance, 2013; Schoenfuss (Personal Communication); Hooi et al., 2004 c. Reason for Measuring: Titratable acidity of the product during processing is measured to ensure the final product will meet manufacturing specifications, as well as parameters outlined by the standard of identity for fruit‐flavored yogurt. d. Principle: A known amount of sample is added to an Erlenmeyer flask along with phenolphthalein indicator and titrated with a standardized solution of 0.1N sodium hydroxide until the sample solution turns a light pink. The total volume of titrant used can be used to calculate total lactic acid in the sample. 4. Brix a. Test: Spindle Method b. Reference: Schoenfuss (Personal Communication); AACCI Method 80‐53.01, c. Reason for Measuring: Brix is measured to ensure that the percentage of total solids from sugar in the product will produce a product with desirable physical and sensory characteristics. d. Principle: See VI.B.17.d

VIII. Tests on Final Product Sampling: Unopened containers of 100g or greater should be randomly selected. Where closed containers are not available, 100g of thoroughly mixed product should be aseptically collected and transferred to a sample container. Sample containers should be shielded from contamination and set on ice. Microbiological analyses, such as coliform count, must be conducted within 24 hours of sample collection. Tests are carried out on the components to follow. 1. Milkfat Content a.Test: Modified Mojonnier Ether Extraction Method b.Reference: Schoenfuss (Personal Communication); AOAC, 2012, Method 989.05 c.Reason for Measuring: Milkfat content of yogurt must meet specifications outlined by the standard of identity and manufacturing specifications. d. Principle: Fat is extracted from yogurt product using a combination of ethyl ether, petroleum ether, ethyl alcohol, and ammonium hydroxide in separate extractions. The extractions are performed in a Mojonnier flask, which is then centrifuged; the ether layer is decanted, and the solvent is boiled off. The final weight of the product is used to calculated percent fat. 2. Total Solids Not Fat a.Test: Solids‐Not‐Fat by Difference between Total Solids and Fat Content b.Reference: Schoenfuss (Personal Communication); AOAC, 2012, Method 990.21 c.Reason for Measuring: Total solids not fat content of yogurt must meet specifications outlined by the standard of identity and manufacturing specifications. d. Principle: Total fat is determined by the modified Mojonnier ether extraction method mentioned above, and total solids are determined by forced draft oven drying method (AOAC Official Method 990.20). The difference between the measured values is representative of total solids not fat in yogurt. 3. Protein a.Test: Kjeldahl Method b.Reference: AOAC, 2012, Method 991.22; Hooi et al., 2004; Schoenfuss (Personal Communication); Chang, 2010 c.Reason for Measuring: Protein may be determined for nutritional labeling of the final product, as well as to meet manufacturer specifications. d. Principle: The yogurt sample is digested using concentrated sulfuric acid in the presence of copper(II) sulfate catalyst, during which nitrogen present in the sample is fixed as ammonium sulfate. The ammonium sulfate is then neutralized into ammonia gas using sodium hydroxide and distilled into a solution of boric acid and methyl red indicator. Titration of the aforementioned solution is carried out with hydrochloric acid until the solution turns a light pink. The total volume of hydrochloric acid titrated can be used to calculated percent nitrogen (because the reactions are stoichiometric), and percent nitrogen can be used in turn to calculate percent protein. 4. Coliform Count a.Test: Petrifilm High‐Sensitivity Coliform Count Plates b.Reference: Schoenfuss (Personal Communication); Hooi et al., 2004; AOAC, 2012, Method 996.02 c.Reason for Measuring: coliform count is measured to determine whether the product is contaminated and either acceptable or unacceptable for consumption. Coliform counts can be an indicator of sanitary packaging practices. d. Principle: A 1:10 dilution of yogurt sample is prepared and brought to a pH of 6.5 to 7.5 with a 1M NaOH solution; 5mL is applied to a 60cm2 area on a PCC plate consisting of 2 plastic films coated in violet red bile nutrients, a dehydrated cold water‐soluble gelling agent, and tetrazolium indicator (a pH indicator). The plate is incubated at 32°C for 24±2 hours. Coliforms produce acid on the PCC plate, which causes the gel to change color, enabling quantitation of cell count. 5. Yeast and Mold Count a.Test: Hydrophobic Grid Membrane Filter (ISO‐GRID) Method Using YM‐11 Agar b.Reference: AOAC, 2012, Method 995.21; Hooi et al., 2004. c.Reason for Measuring: yeast and mold are measured as an indicator of sanitary packaging conditions. The presence of yeast, specifically, in yogurt can reduce shelf life, as it produces gas, which distorts packaging. d. Principle: The sample suspension is administered over a membrane filter that has been treated with hydrophobic residues that prevent the spread of colonies across gridlines. Filters are introduced to a YM‐11 plate and incubated for 50±2 hours at 25±1°C. Squares positive for colonies are enumerated and the number of positive squares is converted to most probable number (MPN) of microorganisms. 6. Viscosity a.Test: (Brookfield Synchro‐lectric) rotational viscometer b.Reference: Tamime and Robinson, 1999; Schoenfuss (Personal Communication) c.Reason for Measuring: The viscosity of yogurt is essential for appropriate sensory characteristics, as well as meeting manufacturer specifications. d. Principle: A rotating spindle rises up and down through yogurt samples. Once the rotational speed and type of spindle are established for a product, precision is high from sample to sample. Accuracy with the rotational viscometer is also high, and risk of local syneresis from the viscometer is low. 7. Lactic Acid/Titratable Acidity a.Test: Spectrophotometric Method b.Reference: Schoenfuss (Personal Communication); Hooi et al., 2004; AOAC, 2012, Method 937.05 c.Reason for Measuring: Measuring lactic acid/titratable acidity is a means of measuring growth of lactic‐acid producing bacteria in the dairy product. d. Principle: Lactic acid is extracted from prepared filtrate of yogurt using ethyl ether. Ethyl ether layer is added to 1cm quartz cell, and the absorbance is taken using a spectrophotometer. The concentration of lactic acid can be determined by comparison to a standard curve. 8. Calcium a.Test: Atomic Absorption Spectrophotometry b.Reference: Hooi et al, 2004; Schoenfuss (Personal Communication); c.Reason for Measuring: Calcium is necessary to measure for nutrition labeling purposes, as well as for physiological importance; manufacturers may also want to know calcium and mineral content to understand how it might affect the physical characteristics and functionality of the yogurt product. d. Principle: samples are prepared for analysis by wet digestion to combust all organic matter. The sample is then sprayed as a mist into a flame, which atomizes it. A hollow cathode lamp excites the calcium atoms, and the change in change in intensity of radiation as a result of the calcium atoms absorbing the energy emitted by the lamp is directly related to the concentration of calcium ions present in the food matrix.

IX. References

AACC International. Approved Methods of Analysis, 11th ed. AACCI International. St. Paul, MN, U.S.A.

AOAC. 2012. Official Methods of Analysis, 19th ed. AOAC International. Gaithersburg, Maryland.

Bradley, Jr. RL. 2010. Moisture and Total Solids Analysis. In: Nielsen SS. Food Analysis. 4th ed. New York: Springer. p. 85‐104.

CFR. 2001. Code of Federal Regulations. Title 21. Part 101.9. Nutritional labeling of food. Office of the Federal Registrar. U.S. Government Printing Office. Washington, D.C.

CFR. 2014. Code of Federal Regulations. Title 21. Part 131.200. Yogurt. Office of the Federal Registrar. U.S. Government Printing Office. Washington, D.C.

Chandan RC, Shahani KM. 1993. Yogurt. In: Hui YH, editor. Dairy science and technology handbook. Volume 2: Product manufacturing. California: VCH Publishers, Inc. p.1‐56.

Chang SKC. 2010. Protein Analysis. In: Nielsen SS. Food Analysis. 4th ed. New York: Springer. p. 117‐132.

Corn Refiners Association. Analytical Methods of the Member Companies of the Corn Refiners Association [Internet]. [Accessed 2014 Dec. 5]. Available from http://www.corn.org/publications/industry‐resources/analytical‐methods/analytical‐methods‐ toc/.

Grade "A" Pasteurized Milk Ordinance, 2013 Revision [Internet]. [Accessed 2014 Nov.]. Available from http://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/milk/ ucm389905.htm.

Hooi R, Barbano DM, Bradley RL, Budde D, Bulthaus M, Chettiar M, Lynch J, and Reddy R. 2004. Chemical and Physical Methods. In: Wehr HM, Frank JF. Standard Methods for the Examination of Dairy Products. 17th ed. Washington, D.C.:American Public Health Association. p.363‐536.

Hoolasi K. 2005. A HAACP study on yoghurt manufacture. Department of Operations & Quality Management, Faculty of Commerce, Durban Institute of Technology.

Malayan Sugar Manufacturing Sugar Specification [Internet]. Accessed [2014 Dec.]. Available from http://www.msm.com.my/sugar‐specification.html.

Milk Quality Improvement Program: Milk Facts [Internet]. Accessed [2014 Oct.]. Available from http://milkfacts.info/.

Modified Starch Farmal™ MS 6125 [Internet]. Accessed [2014 Dec.]. Available from http://www.cosmetic‐specialties.com/docs/MS%206135%20Technical%20Data%20Sheet.pdf.

Oregon Fruit Products Co. Product Data Sheet: Aseptic Seedless Strawberry Puree [Internet]. Accessed [2014 Dec.]. Available from https://shop.fruitforbrewing.com/Content/pdf/Strawberry.pdf.

Schoenfuss, T. 2014. Personal communication. 165 FScN, Department of Food Science and Nutrition, University of Minnesota, MN.

Tamime AY, Robinson RK. 1999. Yoghurt: science and technology. 2nd ed. Cambridge, England: Woodhead Publishing.

TreeTop Fruit Ingredients Product Data Sheet: Aseptic Strawberry Variegate [Internet]. [Accessed 2014 Oct.]. Available from http://foodingredients.treetop.com/documents/Product‐Data‐Sheet‐Fruit‐ Prep‐Aseptic‐Strawberry‐Variegate‐509242.pdf.

USDA National Nutrient Database for Standard Reference, Release 27 [Internet]. [Accessed 2014 Oct.]. Available from http://ndb.nal.usda.gov/ndb/.