31293'1 flifff‘o y W '! Llf‘i'ffi" Mi...2t'é”-" fiat": 1 Us}; Vertig ‘_ ~ A w This is to certify that the dissertation entitled High Performance Liquid Chromatographic Analysis of Natural Fermented Yogurt presented by Michael Lee Richmond has been accepted towards fulfillment of the requirements for Ph. D. degree in Food Science Major professor Date W2 MSU is an Affirmative Action/Equal Opportunity Institution 042771 MSU RETURNING MATERIALS: Place in book drop to remove this checkout from LIBRARIES . ”— your record. FINES W1” be charged if book is I returned after the date “15/ #44/3, stamped below. "" C? Ci "7 (5,1 4:. 1; . --_~C.“ / U” 0 4 2003 £144“, 1 / .. HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF NATURAL FERMENTED YOGURT By Michael Lee Richmond A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition l982 (3(290023'7 ABSTRACT HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF NATURAL FERMENTED YOGURT By Michael Lee Richmond The popularity and consumption of yogurt has increased tremendously in the U.S. A recent study of commercial yogurts revealed wide variation in chemical content, net weight and caloric content. Improved uniformity in composition and quality would benefit yogurt processors. Because phase separation is a concern of commercial importance, experiments were designed to assess the role of secondary packaging and stabilizer blends in reducing physical damage. Stretch wrapping the stack proved effective in minimizing physical damage (P < 0.0l). In another study two high performance liquid chromatographic (HPLC) systems were developed to separate and quantitate various simple sugars and sugar alcohols in food matrices, fresh fruit and yogurt. Using a bonded phase system, two columns connected in tandem and a ternary mobile phase (acetonitrile/water/ethanol) fructose, glucose, sorbitol, sucrose and maltose were accurately separated in twenty minutes. Twenty-four fruits were analyzed for carbohydrate content. Fruits from the Rosaceae family generally contained sorbitol, whereas none of the other fruits examined contained sorbitol. This procedure proved to be fast, simple, and reliable for analyzing simple carbohydrates in food systems, Michael Lee Richmond especially for separating glucose from sorbitol. This was accomplished by addition of a second column. A majority of the world's population is considered lactose intoler- ant. Because lactose occurs naturally in many dairy foods, and is added commercially to a variety of non-dairy foods in the form of lactose, dried whey or milk, there are concerns about lactose content in many foods. An HPLC procedure was developed to separate and quantitate lac- tose, glucose and galactose in dairy products. The system contained a temperature elevated resin-based column (80°C) and used water as the eleuent. The three sugar mixture was easily separated in ten minutes. Hydrolysis of lactose and accumulation of glucose and galactose was fol- lowed through ripening and long term storage of yogurt. Lactose content decreased from 7.l2% to 4.l9% after l4 days, while galactose content increased from 0% to 1.06% at l4 days. Glucose remained at trace levels. Autoclaved lactose containing microbiological media were also evaluated using this system.' A compound having the same retention time as lactu- lose was observed in autoclaved media. I wish to dedicate this book to my parents Lester and Josephine. Mom and Dad I love you both. Thank you for your support and encour- agement. I could not have done it without you. ii ACKNOWLEDGEMENTS I would like to thank my committee members Drs. Bruce Harte, Hans Lillevick, J. R. Brunner, J. I. Gray, and especially my advisor Dr. Charles M. Stine for staying with me through the years -- they taught me alot. I would like also to thank my family, in particular, my wife Eileen for letting me go in all those nights to work. Thanks go also to my daughters Lisa, Bethany and Sarah. I am also indebted to the University of Guelph for preparing and evaluating taste panel data, as well as Dr. Ramesh Chandan for helping in this endeavor. I also want to thank Sterling Thompson for performing some microbiological analyses. Others who helped and I wish to acknow- ledge are: Dr. J..Gill, Dr. H. R. Struck, Dr. C. J. Mackson. Numerous students also helped me with various experiments and projects to which I am deeply indebted; they include Dave Barfuss, Maxwell Sherman, John Stevenson and Dave Staleyu In addition, I would like to thank the secretaries that I have worked with over the years -- Debby, Tammy, Patti and Mary. Finally, I would like to thank those who have helped me along the way and I have forgotten to mention here -- thank you all, I appreciated it. iii TABLE OF CONTENTS LIST OF TABLES . . . . . LIST OF FIGURES . . . . . . . INTRODUCTION . . . . . . . . . . . . CHAPTER I - INTRODUCTION AND REVIEW REVIEW OF THE LITERATURE . . . . . . Nutrition Information . . . . . . . Attitudes, Marketing, Sales . . . . Regulations, Quality, Composition. Processing and Manufacturing Concerns Protein, Carbohydrate, Fat Stabilizers . . . . . Heat Treatment . . . Yogurt Cultures . . . Culture Enumeration . Culture Activity . . Flavor of Natural Yogurt Fruit Addition . . . . Sensory Evaluation Score Cards Storage and Packaging . . . . . Yogurt Products . . . . ...... Literature Cited . . . . . . . CHAPTER II - YOGURT, A COMPOSITIONAL LANSING AREA 0 O O O 0 O O O O 0 O SURVEY IN Introduction . . . . . . . . . . . Materials and Methods . . . . . . . Results and Discussion CHAPTER III - PRODUCTION, PROCESSING AND YOGURT OF SWISS STYLE Introduction . . . . . Materials and Methods . Results and Discussion Summary . . . . . . . . Literature Cited . . . Literature Cited . . . . . . . . . HONEY O 0 O O 0 iv 0 O O O O O O 0 SENSORY O O O 0 O O O O O 0 0 O O O O 0 O O O 0 THE GREATER O O O O 0 O O O O O O O O 0 O 0 EVALUATION 0 O O O O O O O O O O O O O 0 Page vi ix 104 105 107 109 116 117 118 120 124 130 131 Page CHAPTER IV - PHYSICAL DAMAGE OF YOGURT, THE ROLE OF SECONDARY PACKAGING ON STABILITY OF YOGURT . . 132 Introduction . . . . . . . . . . . . . . . . . . . . . 133 Materials and Methods ........ . . . . . . . 134 Results and Discussion . . . . . . . . . . . . . . . . 138 Conclusions . . . ...... . . . . . . . . . . . 144 Literature Cited . . . . . . . . . . . . . . . . . . . 145 CHAPTER V - SEPARATION AND ANALYSIS OF CARBOHYDRATES IN YOGURT AND FRESH FRUIT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ................ 146 A. SEPARATION OF CARBOHYDRATES IN DAIRY PRODUCTS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Introduction . . . ...... . ...... . 147 Materials and Methods . . . . . . . . . . . . . 150 Results and Discussion . . . . . . . . . . . . 153 Literature Cited 0 I O O O O O O O O O O O O O 164 B. SEPARATION AND QUANTITATION OF CARBOHYDRATES IN LOWFAT PLAIN YOGURT AND LACTOSE CONTAINING MICROBIOLOGICAL MEDIA Introduction . . . . . . . . . . . . . . . . 167 Materials and Methods . . . . . . . . . . 168 Results and Discussion .. . . . . . . . . . . 174 Literature Cited . . . .g. . . . . . . . . . . 190 C. ANALYSIS OF SIMPLE SUGARS AND SORBITOL IN FRUIT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY . - - Introduction . . . . . . . . . . . . . . . . . 192 Experimenta] O O I O O O O O O 0 O O O O O O O 195 Results and Discussion . ..... . . . . . . 197 Literature Cited . . . . . . . . . . . . . . . 205 CHAPTER VI - SUMMARY . . . . . . . . . . . . ..... . . 206 APPENDICES Appendix I . . . . . . . . . . . . . . . . . . . . . . 210 Appendix II ..... . . . . . . . . . . . . . . . . 214 LIST OF TABLES CHAPTER I Table Page 1. Nutrient content of commercial yogurts . . . . . . . . 5 2. Chemical composition of various flavored yogurts . . . 13 3. Qualities of an ideal yogurt starter . . . . . . . . . 23 4. Acetaldehyde content and acidity of various yogurts . 53 5. Compounds in yogurt aroma . . . . . . . . . . . . . . 60 CHAPTER II Table Page 1. Chemical composition of various brands of lowfat flavored yogurt . . . . . . . . . . . . . . . . . . . 110 Chemical composition of various flavored yogurts . . . 110 Chemical composition of various brands of plain yogurt . . . . . . . . . . . . . . . . . . . . . . . . 112 Net weight of various brands of flavored lowfat commercial yogurts . . . . . . . . . . . . . . . . . . 114 . Calculated caloric content of various commercial yogurts . . . . . . . . . . . . . . . . . . . . . . . 115 0'1 «rfi (JON 0 CHAPTER III Table Page 1. Biochemical tests to aid in identification of yogurt culture bacteria . . . . . . . . . . . . . . . . . . . 125 2. Initial evaluation of buckwheat honey addition to lowfat plain yogurt . . . . . . . . . . . . . . . . . 126 3. Effect of buckwheat honey added to yogurt mix before (BP) and after pasteurization (AP) as related to set 12 time . . . . . . . . . . . . . . . . . . . . . . . . . 9 vi CHAPTER IV Table Page 1. Characteristics of secondary packaging materials for shipping yogurts . . . . . . . . . . . . . . . . . . . 136 2. Statistical analysis of physical damage occuring in yogurt vibrated in selected shippers . . . . . . . . . 140 CHAPTER V SEPARATION AND QUANTITATION OF CARBOHYDRATES IN LOWFAT PLAIN YOGURT AND LACTOSE CONTAINING MICROBIOLOGICAL MEDIA Table Page 1. Statistical data: regression equations and correla- tion coefficients for lactose, glucose and galactose standard curves . . . . . . . . . . . . . . . . . . . 172 2. Recovery of lactose and galactose from a spiked sample of lowfat plain yogurt . . . . . . . . . . . . 173 3. pH, lactose content and galactose content of lowfat plain yogurt mix and yogurt during ripening and storage . . . . . . . . . . . . . . . . . . . . . . 181 4. HPLC analysis of lactose content (%) of various lactose containing microbiological media . . . . . . . 185 ANALYSIS OF SIMPLE SUGARS AND SORBITOL IN FRUIT BY HIGH PER- FORMANCE LIQUID CHROMATOGRAPHY Table ' Page 1. Linear re ression equations and correlation coeffi- cients (r for carbohydrate standards . . . . . . . . 199 2. HPLC analysis of simple sugars in some common fruits . 201 3. HPLC analysis of simple sugars and sorbitol in fruits of the Rosaceae family . . . . . . . . . . . . . . . . 202 APPENDICES Appendix I Table Page .1. Analysis of variance table for color . . . . . . . . . 210 2. Analysis of variance table for sweetness . . . . . . . 211 3. Analysis of variance table for texture . . . . . . . . 211 4. Analysis of variance table for flavor . . . . . 212 5. Analysis of variance table for overall acceptability . 212 6. Sample means for each attribute . . . . . . . . . . . 213 vii Appendix II Table Page 1. Honey yogurt score card . . . . . . . . . . . . . . . 214 viii LIST OF FIGURES CHAPTER I Figure . ' Page 1. Flow diagram of yogurt manufacture . . . . . . . . . . 18 2. HPLC chromatogram of strawberry yogurt (1) sucrose (2) Lactose (3) glucose (4) galactose (5) fructose. Bio-rad HPX-87 carbohydrate column (80°C); Aminex A-25 Micro-guard Anion/OH cartridge; solvent, H20; flow rate, 0.6 ml/min.; injection volume, 4ul; attenuation 2 8X . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3. Dependence of shelf life of cultured milk on the technological process . . . . . . . . . . . . . . . . 38 4. Inhibitory effect of various sugar additions on acid development of a yogurt culture . . . . . . . . . . . 44 5. Acetaldehyde production by a single strain of S, ther- mo hilus, L, bulgaricus and a 1:1 mixture of both . . 57 6. Flow sheet diagram for frozen yogurt manufacture . . . 72 CHAPTER III Figure Page 1. Yogurt processing scheme . . . . . . . . . . . . . . . 123 CHAPTER IV Figure Page 1. MTS electrohydraulic vibration table . . . . . . . . . 137 2. Comparison of shipper types and related physical damage in lowfat plain yogurt following vibration . . 141 ix CHAPTER V SEPARATION OF CARBOHYDRATES IN DAIRY PRODUCTS BY HIGH PERFORMANCE LIQUID CHORMATOGRAPHY Figure Page 1. HPLC chromatogram of standard carbohydrate solution (1) lactose (2) glucose (3) galactose. Bio-Rad HPX-87 carbohydrate column (80°C); Aminex A-25 Micro- guard Anion/0H cartridge; solvent, H20; flow rate, 1.0 ml/min; injection volume, 4u1; attenuation, 8X . . 152 2. HPLC chromatogram of standard carbohydrate solution (1) sucrose (2) lactose (3) glucose (4) alactose. Bio-Rad HPX-87 carbohydrate column (800C); Aminex A-25 Micro-guard Anion/0H cartridge; solvent, H20; flow rate, 1.0 ml/min; injection volume, 2.5u1; attenuation, 8X . . . . . . . . . . . . . . . . . . . 154 3. HPLC chromatogram of standard carbohydrate solution (1) sucrose (2) lactose (3) glucose (4) alactose. Bio-Rad HPX-87 carbohydrate column (BOOCI; Aminex A-25 Micro-guard Anion/0H cartridge; solvent, H20; flow rate, 0.6 ml/min; injection, 2.5u1; attenuation, 8X . . . . . . . . . . . . . . . . . . . 155 4. HPLC chromatogram of strawberry yogurt (1) sucrose (2) lactose (3) glucose (4) galactose (S) fructose. Bio-Rad HPX-87 carbohydrate column (80°C); solvent, H20; flow rate, 0.6 ml/min; injection volume, 4u1; attenuation, 8X . . . . . . . . . . . . . . . . . . . 157 5. HPLC chromatogram of strawberry yogurt (1) sucrose (2) lactose (3) glucose (4) galactose (5) fructose. Bio-Rad HPX-87 carbohydrate column (80°C); Aminex A-25 Micro-guard Anion/0H cartridge; solvent, H20; flow rate, 0.6 ml/min; injection volume, 4ul; attenuation, 8X . . . . . .1. . . . . . . . . . . . . 158 6. HPLC chromatogram of cultured buttermilk (1) lactose. Bio-Rad HPX-87 carbohydrate column (800C); Aminex A-25 Micro-guard Anion/OH cartridge; solvent, H20; flow rate, 0.6 ml/min; injection volume, 2.5ul; attenuation, 8X . . . . . . . . . . . . . . . . . . . 161 CHAPTER V (Continued) SEPARATION AND QUANTITATION OF CARBOHYDRATES IN LOWFAT PLAIN YOGURT AND LACTOSE CONTAINING MICROBIOLOGICAL MEDIA Figure Page 1. Progressive HPLC chromatograms of carbohydrate extracts from yogurt mix (1.5% fat, 12.6% SNF) and yogurt. Conditions: Aminex HPX-87 carbohydrate col- umn maintained at 80°C; Bio-Rad Aminex Anion/0H MicroguardTM cartridge. Refractive index detector; attenuation, 8X; flow rate, 0.6 ml/min; solvent, reverse osmosis ion-exchanged water~. . . . . . . . . 176 A. Carbohydrate extract from unheated lowfat plain yogurt mix before heat treatment; large peak lactose. Zul injection volume; 50/50 dilution with ROIE water . . . . . . . . . . . . . . . . . 177 B. Carbohydrate extract from heated lowfat plain yogurt mix (88°C, 40 min.); large peak lactose. 2 pl injection volume; 50/50 dilution with ROIE water . . . . . . . . . . . . . . . . . . . . . . 178 C. Carbohydrate extract from lowfat plain yogurt after ripening to pH 4.6. Large peak lactose, next peak galactose. 2u1 injection volume; 50/50 dilution with ROIE water . . . . . . . . . . . . 179 D. Carbohydrate extract from lowfat plain yogurt after ripening and storage. Large peak lactose, next peak glucose and last peak galactose. Zul injection volume; no dilution . . . . . . . . . . 180 2. HPLC chromatograms of carbohydrate extract from litmus milk medium. Conditions: Aminex HPX-87 carbohydrate column maintained at 80°C; Bio-Rad Anion/0H Micro- guardTM cartridge. Refractive index detector; atten- uation, 8X; flow rate, 0.6 ml/min; solvent, reverse osmosis ion-exchanged water . . . . . . . . . . . . . 186 A. Carbohydrate extract from non-autoclaved litmus ‘ milk, 2“] InjeCt‘ion O O O O O O O I O O O O O O O 187 B. Carbohydrate extract from autoclaved litmus milk (121°C, 15 min.), 2 ul injection . . . . . . . . 188 3. HPLC chromatogram of standard lactose (0.50% wt/vol.) and lactulose (0.25% wt/vol.). Large peak lactose, next peak lactulose. 4ul injection; flow rate, 0.4 m1/min. HPX-87 carbohydrate column (80°C) and Bio-Rad Anion/0H MicroguardTM column. Refractive index; attenuation, 8X . . . . . . . . . . . . . . . 189 xi CHAPTER V (Continued) ANALYSIS OF SIMPLE SUGARS AND SORBITOL IN FRUIT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Figure 1. Page HPLC chromatogram of standard carbohydrate mixture. Dual column arrangement; mobile phase, acetonitrile/ water/ethanol (80/15/5; v/v/v); flow rate, 1.8 ml/min; injection volume 10u1; attenuation, 8X . . . . . . . . 198 HPLC chromatogram of carbohydrates in the orange. Dual column arrangement; mobile phase, acetonitrile/ water/ethanol (80/15/5; v/v/v); flow rate, 1.8 ml/min; injection volume, Sul; attenuation, 8X . . . . . . . . 203 HPLC chromatogram of carbohydrates and sorbitol in the purple plum. Dual column arrangement; mobile phase; acetonitrile/water/ethanol (80/15/5; v/v/v); flow rate, 1.8 ml/min; injection volume, Sul; attenuation, 8X . . . . . . . . . . . . . . . . . . . . . . . . . . 204 xii INTRODUCTION In recent years, the popularity of yogurt in the U.S. has grown tre- mendously. While many factors are responsible for the expansion of this market, fruit addition along with multimedia advertising are considered most important. Because of this sudden growth, a definite need has arisen for broad range scientific research of yogurt. Several important areas needing further examination are product com- position and development, physical and chemical damage during distribution and storage, current literature status and various processing parameters. Lactose intolerance reportedly affects a large portion of the world's population. Dairy products play a vital role in the diets of many indi- viduals and because lactose is often in moderate to high concentrations in various dairy products such as yogurt, rapid and accurate measurements of lactose and other sugars are needed for industrial and academic pur- poses. The separation ana analysis of simple sugars and sugar derivatives, whether added to foods commercially or occurring naturally, is a current and important nutritional topic. This presentation addresses these concenrs and outlines them in chapter form with the intent of (1) providing a broad spectrum of prac- tical research about yogurt, and (2) providing information on the separa- tion and analysis of simple carbohydrates in dairy products and (simple sugars and sorbitol) in various botanical families of fruit. CHAPTER I INTRODUCTION AND REVIEW] 1A Review of Yogurt Technology with Emphasis on Research Since 1970. To be submitted to Dairy Sci. Absts. (England) REVIEW OF THE LITERATURE Yoghourt, Yoghurt, Yogurt. There seem to be as many names for this food as there are flavors. The popularity of this nutritionally healthful food has increased the world over in recent years. Although per capita consumption in the U.S. is well below that of most European countries, significant increases have been made during the past decade. The popularity of yogurt has increased for many reasons. However, fruit and other flavors, increased advertising expenditures and marketing strategies have played an important role in developing the current demand for this excellent food. Consumption in the U.S. is currently about 1.2 kg per capita compared to 2.26 kg per capita for cultured buttermilk (Milk Ind. Found., 1981). European consumption rates for yogurt are much higher than U.S. figures (Rasic and Kurmann, 1978). Increasing consumption of yogurt in the United States by improving marketing strategies and product quality will benefit the dairy industry and other related industries (fruit, preserves, packaging) as well. This review of yogurt will be primarily concerned with recent advances in production and research since the review by Humphreys and Plunkett (1969). This paper will primarily discuss yogurt made from bovine milk using the mixed starter culture Lactobacillus bulgaricus and Streptococcus thermophilus. Further, since previous reviews have focused on yogurt manufacture outside the Continental U.S. (Europe and Australia) this review will highlight yogurt manufacture and current research in the U.S. and abroad. 4 Since 1969 a number of reviews describing various aspects of yogurt have been published (Humphreys and Plunkett, 1969; Lundstedt, 1971; Mann, 1973 a,b,c; Robinson and Tamime 1975, Kosikowski, 1977; Tamime and Deeth, 1980). Rasic and Kurmann (1978) recently published a book on yogurt, and various chapters have also been devoted to this subject (Kosikowski, 1977; Chandan, 1982). An interesting article on the origin and culture of fermented milks, including yogurt was described by James (1975). In 1969, Humphreys and Plunkett reviewed yogurt and its manufacture describing many processing parameters, and Lundstedt (1971) discussed manufacture with regard to incubation temperature, suggesting a lower incubation temperature (30°C) and longer incubation time (12-16 h) would improve the product. Mann (1974) updated literature from around the world, discussing many concerns about yogurt in a three-part series (l973a,b,c). In 1975, Davis described developments in yogurt technology in the United Kingdom (U.K.). He reported on the history of fermented milks, the role of yogurt bacteria, and also described a process for continuous manufacture of yogurt. Robinson and Tamime's review in 1975 outlined numerous methods used world wide for the production of yogurt; methods for processing stirred, set and liquid yogurt and their respective processing parameters were described. More recently, Tamime and Deeth (1980), in an exhaustive review, covered the technology and biochemistry of yogurt. They discussed physical and chemical changes during processing, fermentation and storage. Aa~m_v __u==ou »2.aa .aco.aaza Illiill a. N_ c~e. o “no.8...ema_wo_ no.5o. so. an RN_ ao_ m. co. an. ma. «0.». . We. Nn. «a. m_.~ mad m_. amnzwms .occ_. a Emmnghwflouu.ntm a 2 an. 2 3932248.??? 8. a... as m: 2 84:8; 8a m 3. a. 8. m2 8.: an 3 8.2 £82.32 a : fiwmb a NNN ..__.=~> ecu maccmu N 22o. N. 393.3%.”1838. a. 2 mm a: 2 8. 8.12;: ~ g. 8. 2. r1 2a 5 a... US 38: a magpwmfiwafl 8 2 $9 : $589232... :38. 2.. 2:2 3. 2 8. a: m: z: o 8. 2. 8.. 34:38 2 8.8— 38: o zwfiwmmicwam r: em Na. N NS 2:. 2: N: a: 8.? 2 a: 3 2 B. a. 2. a: 2 8. 3 S.~ «~23 R 8 Si :82... ammmfluwmm . r11 . .tsoo» AAASAdNUlVZdeNIO DJdSWJSl In. I? I? o I} a I... Is. u. s I? O o E to ‘ a! o unnunwnmumwwnmwmn munnwnmn wmmpwwmomm mac» 1 namxmxu... uuuuum “Hum INIMM IWSVSVflO vvramaam..m Mmmm w nymouup 9 u u 5 V S . 0 Ph. P .n T. M V D. .M .w Sim .DM .0 11 “1 no mu .m p .w A H V 3 11 .0 Va 3 H l l. S D. l. 1. nl U D. r. w u .. w m ) u a m u 6 m p a Va 6 J U l u‘ s I? m K 6 s s “w u 1. x. N .N X r... a x t 1 $3393» b .9 r. 3.8.8.89 .6 23:3 325:: mm mm _ usm Omccgm ._ 0030.0 Hi 05.000 L 1.1 . a 00..%:00.i_ Hi 0503.0... i1— __. 10.000. 0.00.: . h¢38> hum aiiihiiilillulliv fl 05.30 0000.0 5.! 05.0.0000. H ._ 05.900000 05.30 fl 0.0.8000... 00:00:00. 0. .oooL J ._. —1 .6035003 .00: i— 4 —1 002050000000... _ .— 3.00.0 .0250... 0055.0... _ h 19 Tamime and Robinson (1978) described the production of concentrated yogurt (labneh) traditionally consumed in the Middle East. Product concentration is achieved by putting the yogurt into a cloth bag and hanging it to allow whey seepage, or by stacking or piling bags on top of each other. Because of the popularity of this product and the desire to improve final product uniformity, labneh production was evaluated to determine factors influencing quality. Natural yogurt (16% T.S.) proved to be a good starting point for labneh production (24% T.S.). Products of solid contents less than 20%, and greater than 25%, were found to be poorer in quality. Various cultures were used to manufacture labneh. In another article (Tamime, 1978) the production of yogurt and concentrated yogurt from hydrolyzed milk was discussed. A commercial neutral yeast lactase was used to hydrolyze the lactose. In this study 50% hydrolysis (35°C) was achieved in 45 min. and 99% in 4 h. Tamime reported processing time could be reduced by up to 1 h using hydrolyzed milk. Increased starter activity was related to increased free glucose in the system. By using hydrolyzed milk to make concentrated yogurt, a sweeter product was produced. Starters played an important role in the drainage time of whey. A slime-forming culture (RR) showed the least amount of extracted whey. This culture was not desirable in concentrated yogurt production, since the product became very gummy and lost some of its traditional character. Jepsen (1977) described the use of membrane filtration for the manufacture of yogurt. Using ultrafiltration (UF) membranes, 20 protein content was increased without increasing lactose content. Another advantage of UF is the ease with which it can be incorporated into a continuous system. Veinoglou (1978) discussed the production of strained yogurt from UF milk. For production purposes, best results were obtained with 8.5% protein and 9.5% fat. Sensory properties were similar to the traditional product. Nielson (1976) reported on the use of whey solids in yogurt. Dry whey (0.2 - 0.6%) increased viscosity and enhanced acid development. Advantages of using whey and dry whey in yogurt were discussed. Macbean (1978) discussed the development of mechanized and continuous methods for the production of yogurt. Yogurt mix was heated to 92°C, 45 min. and a two-stage fermentation system used. Yogurt mix was incubated to pH 5.5 (45°C) in a stirred fermenter and pH controlled by inflow of unfermented mix. Mix from the first stage moved into the second stage where it was continued to ferment and allowed to coagulate in a tubular flow fermenter as the mix moved slowly downwards at controlled temperature ( <45°C). Difficulty in achieving tubular flow in the pilot scale second stage tubular-flow-fermenter was noted resulting in yogurt with poor texture. In another article, Macbean gt 31 (1979) described pH-stat continuous cultivation and stability of mixed fermentation in continuous yogurt production. The general use of continuous fermentation in yogurt manufacture was discussed. Angevine (1972) reported on processing yogurt and acidophilus yogurt. He reported long set yogurt (30-32°C; 12-16 h) resulted 21 in better balance of yogurt bacteria producing more uniform flavor. A procedure for processing acidophilus yogurt was also described. Also discussed were nutritional incentives ascribed to acidophilus yogurt. Mann (1978) reported that acidophilus milk was rapidly becoming a product of commercial importance in the U.S., although production figures do not support this very well. Currently availability of this product as well as liquid yogurt is primarily concentrated in "health food stores" and often at high cost. Rasic and Kurmann (1978) described processing and manufacture of various cultured milks in their book. While there are many patents involving cultured dairy products, Igoe (1979) described an interesting patent for direct acidified yogurt. The yogurt was prepared from milk, a thickener blend (starch and various vegetable stabilizers) and food grade acidulant. After pasteurization and acidification the mix was subjected to a shearing treatment to produce yogurt like texture. A commercial yogurt currently being introduced in the U.S. is made from goats milk. Aggarwal (1974) discussed manufacture of goats milk yogurt. Yogurt mix (goats milk, 4.3% fat) was pasteurized (88°C, 30 min) but not homogenized. Acid development was faster in goat milk than bovine. Goat milk yogurt was whiter in color due to a lack of carotenoid pigments, and the fat globule size. Eventhough goats milk yogurt was not homogenized, there was no detectable fat or cream line due to the smaller fat globules in such milk. Aggarwal reported that acetaldehyde production masked the typical “goaty'I flavor. This yogurt did not show any signs of phase separation. Ouitschaever (1978) described the manufacture of yogurt 22 11,21 '1' u L O 5 10 15 TIME mm.) ’ Figure 2. HPLC chromato ram of strawberry yogurt: (1) sucrose, (2) lactose, C3) glucose, (4) galactose, (5) fructose. Bio-rad HPX-87 carbohydrate column (80°C); Aminex A-25 Micro-guard Anion/0H cartridge; solvent, H20; flow rate, 0.6 ml/min.; Injection volume, 401; attenuation, 8X. 23 TABLE 3 Qualities of an ideal yogurt starter.a 1 Purity, i.e., free from contaminants. 2 Vigorous growth. 3 Production of the right consistency. 4 Production of a good flavor without off flavors. 5 Stability, i.e., its balance should be easily maintained. 6 No tendency to induce syneresis. 7 Should not develop excessive acidity on cold storage. 8 Should have a reasonable tolerance to sugar. 9 Should be resistant to penicilin and other antibiotics. 10 Its maintenance should be easy. 11 It should be phage resistant. aTramer (1973) 24 from goat and bovine milk. Goat milk was standardized to 2.0% fat, homogenized at zookg/cm2 (2800 psi), pasteurized at 80°C, 15 min, cooled to 45°C and inoculated with culture. Goat milk was fortified with 4% goat skim milk powder; a pH of 4.5 was attained in 170 min. The yogurt did not whey-off during storage at 4°C and was well liked, especially when sugar and/0r flavorings were added. In a series of articles Pinthong 3L El (1980 a,b,c) described the development of a soy-based yogurt product. During development a product with acceptable acidity was produced using soy milk, 1.0% glucose and 0.1% yeast extract; glucose increased acid production and yeast extract was incorporated to stimulate L, bulgaricus (Pinthong 2L.al, 1980a). After incubation a firm homogenous curd was produced. Phase separation was minimal. In a second article (Pinthong, 19800) various systems (soy milk +‘§. thermophilus; soy. milk + L, bulgaricus; soy milk + mixed culture) were used to produce a yogurt-like product. Compounds detected included acetaldehyde, acetone, methanol, ethanol, n-pentanal and n-hexanal. Sensory properties were related to levels of n-pentanal produced by S. thermophilus and n-hexanal naturally present in the soy milk. Pinthong 2L.al (19800) described the effect of fermentation (using various bacteria) on the levels of oligosaccharides present. High performance liquid chromatography (HPLC) was used for carbohydrate analysis. The degree of oligosaccharide removal was small however. L, bulgaricus reduced the beany odor by removing some of the n-hexanal (Pinthong, 1980c). Richmond 33 11 (1982) used HPLC to separate sugars in strawberry yogurt (Figure 2). Milk salts were present in the area before the sugar peaks. Using a 25 resin-based LC system lactose and sucrose were not adequately resolved but excellent resolution between isomers, glucose and galactose is shown in this figure. Protein, Carbohydrate, and Fat Baysu (1972) investigated changes in amino acid, protein and lactose content of milk during fermentation. Mean values for amino acid content in 25 yogurt samples were reported. Mean values for total protein in milk and yogurt samples were 3.38 and 3.39%, respectively. The decrease in arginine content (milk to yogurt) was significant at the 5% level. Lactose content and pH were significantly different at the 1% level, which was not surprising. Schalichev 35 31 (1971) determined free amino acids in raw milk, heat-treated milk and yogurt. They reported accumulation of free amino acids in yogurt was dependent on heat treatment of the raw milk. The quantity of free amino acids increased with an increase in heat treatment. ' Free amino acids in cows milk and yogurt (24 and 48 h after production) were qualitatively and quantitatively determined by Kopac-Parkaceva 3L.al (1975). Seventeen amino acids in raw milk and yogurt were identified but their content in yogurt varied depending on the kinds and ratios of starter used. One- day-old yogurt (1:1) showed an increase in all free amino acids, especially tyrosine, phenylalanine and leucine. When starter ratio was changed (1:3; L bulgaricus: S, thermophilus) proline was the major amino acid present. In two-day-old yogurt (1:1) valine, 26 phenylalanine, lysine and leucine were in greatest concentration. They reported that yogurt produced with a starter ratio of 1:1 contained larger amounts of free essential amino acids. El-Shibiny 35.31 (1979) discussed the effect of storage on the proteins of zabadi at refrigerator temperatures. Zabadi was periodically analyzed for total nitrogen, non-protein nitrogen, total free amino acids, soluble tyrosine and tryptophan. During storage non-protein nitrogen, soluble tyrosine and free amino acids increased but storage had no effect on the electrophoretic pattern of zabadi proteins. In a related article these same authors (El-Shibiny‘gL EL, 1979) discussed the effect of storage on various chemical properties of zabadi. During storage total solids decreased as a result of lactose hydrolysis. Fat content decreased slightly during storage. The acidity of fresh samples increased and pH decreased with increasing concentration of dry milk used. Total volatile fatty acids increased during 7 days storage. Acetaldehyde increased throughout storage as did glucose and galactose content. Groux (1973), in a discussion of yogurt aroma, reported aromatic components found in yogurt may be from enzymatic deamination of certain free amino acids. He also reported that modified milk proteins resulting from bacterial action were important for coagulum structure. Formisano SE 31 (1971a) discussed the proteolytic activity of L, bulgaricus and S, thermophilus in yogurt. They further discussed the role of the cooling phase on the free amino acid content of yogurt. Both organisms possessed a caseinolytic 27 enzyme system. Luca (1972) studied the decomposition of non-casein proteins by lactic acid bacteria (LAB) in yogurt manufacture. He reported L, bulgaricus was more proteolytic than S. thenmophilus and when both organisms were inoculated together, reduced proteolysis was noted. In general, nitrogen content of the total albumin and globulin fraction decreased during the first two days and then increased. Increases in the nitogen content of the proteose-peptone fraction was also observed. Formisano 22.21 (1974) reported the degree of proteolysis was limited during incubation and the resulting amino acid pool was characterized as having 21 amino acids and seven ninhydrin-positive substances not identified. Predomi- nant amino acids included glutamic acid, proline, serine, x-alanine and aspartic acid. During storage they observed a decrease in neutral fat, while free fatty acid content increased. Further, fatty acids with higher carbon number (C14:0 to °l8:2) were more numerous than those containing fewer carbon atoms (C4-C12). Terplan SE 31 (1973) evaluated 23 micro-organisms for their ability to produce histamine and tyramine in yogurt. Over one-half the organisms were able to form histamine; however, bacteria with decarboxylase activity were able also to reduce amines. No health concerns were apparent from the amounts of histamine and tyramine produced. Popov and Zakhariev (1973) followed hydrolysis of lactose in Bulgarian sour milk using paper chromatography (PC). They found only a small amount of lactose was hydrolyzed; this was explained by 28 the weak ES-galactosidase activity of the culture. Lee and Lillibridge (1976) described an ascending thin layer chromatographic (TLC) procedure for determining lactose in various foods including yogurt. Sample preparation and analysis time was quite long. Goodenough and Kleyn (1976) used TLC to follow hydrolysis of lactose during ripening of yogurt. They reported about one-third of the lactose was hydrolyzed during incubation. Mouillet 2L.gl (1977), using a GLC procedure, found 35% of original lactose was hydrolyzed during incubation, glucose was used up by the LAB and galactose accumulated during ripening. These results agree closely with Goodenough and Kleyn (1976). Recently, HPLC has been used to determine lactose and other carbohdyrates in dairy products (Waters Assoc., 1978; Euber and Brunner, 1979; Richmond 21 31, 1982). Both bonded phase and resin based liquid chromatographic (LC) systems have proved useful in separation and quantitation of sugars in dairy products (Figure 2). Samples are easily prepared and analysis times are generally less than 15 min. Dean (1978) used the Technicon Auto Analyzer to determine free sugars in yogurt. A nut yogurt contained 9.7% sucrose, 3.2% lactose, 1.0% fructose, 1.3% galactose and 0.9% glucose. Washuttl SE 31 (1973) reported on the content of sugar alcohols in foods. The only sugar alcohol found in yogurt was galactitol at 893 mg/100 g. Engel (1973) used a commerical lactase preparation (Maxilact) to modify yogurt. He reported that yogurt with 50% lactose hydrolysis would be an acceptable product that was sweeter than natural yogurt 29 without increasing caloric content. Gyuricsek and Thompson (1976) used a commercial yeast lactase to obtain 90% hydrolysis of yogurt milk. Yogurt culture was added and the mix incubated to pH 4.6. Reported advantages included reduced incubation time (40 min decrease). Tartness was decreased and glucose content increased due to hydrolysis. Sensory evaluations indicated that hydrolyzed lactose yogurt to be an acceptable product. No discussion of milk sugar is complete without mentioning lactose intolerance. Because dairy products are an important part of our calcium intake, Gallagher 25.21 (1975) studied three lactase- deficient patients and found they tolerated fermented dairy products without symptoms of this malady. In all three subjects fecal calcium paralleled increased lactose intolerance symptoms. Results indicated that calcium absorption improved when consumed in fermented dairy foods. Hurt (1972) reported that because milk and other dairy products are often regarded as staples in the diet, there is concern by some segments of the food industry regarding addition of whey and NFDM to foods because of their lactose content. Hurt recognized that lactase deficient individuals could obtain dairy food nutrition by consuming fermented products rather than fresh dairy foods. Escobar and Guillot (1974) described the use of yogurt and cheeses in the treatment of patients with lactose malabsorption. When milk was withdrawn from the diet (49 patients) yogurt administration was considered favorable in 76% of the patients. The value of these milk substitutes was emphasized for those requiring increased protein, calories and calcium in the diet. 3O Goodenough and Kleyn (1976) fed laboratory rats yogurt, pasteurized yogurt and simulated yogurt with sucrose or lactose for 7 d. Assays of blood galactose demonstrated that animals fed natural yogurt (containing viable bacteria) were able to absorb galactose more efficiently. Castro-intestinal survival of culture organisms was demonstrated 12.2122 up to three hours after feeding. Hargrove and Alford (1978) observed growth rate and feed efficiency in rats fed yogurt and other fermented milks. Fermented products tested were yogurt, three types of acidophilus milk, lactic buttermilk, Bulgarian buttermilk and direct acidified milk. In six different trials yogurt gave better weight gains than other milks. Even though L, bulgaricus was found in the intestinal tract during feeding trials, it disappeared after 3 d when no longer fed. .S. thermophilus was never isolated below the upper small intestine, but L, acidophilus was usually present and persisted when no longer in the diet. Recently in the U.S. more emphasis is being placed on the milk solids nonfat (MSNF) fraction of milk products. Graf (1975) reported that low fat dairy products were selling surprisingly well. He discussed market implications of changing the fat content in milk and dairy products. The contribution of fat to the flavor ' of dairy products is well known. Because much of the yogurt consumed is low fat and because of the low lipolytic activity of yogurt bacteria, flavor contributions are considered minimal. Bills 33 El (1969) reported objective laboratory analysis and sensory evaluation has provided insight into the role of free fatty acids as flavor compounds in dairy products. They discussed the importance 31 of short chain fatty acids including acetic acid, and pH on flavor, when pH of the medium is below 4.5 undesirable acetic acid or vinegary flavors may be observed. Formisano EL 21 (1971) used gas chromatography (GC) to describe variations in free and esterified fatty acids in fermented milks and ratios between saturated and monounsaturated fatty acids. Differences in the metabolic behavior between the two yogurt bacteria were also discussed. Rasic and Vucurovic (1973) studied free fatty acid content of yogurt made from various milks. In cow's milk, saturated fatty acids generally increased when compared to initial milk except stearic acid which decreased. Oleic, linoleic and palmitoleic acid decreased. In ewe's milk yogurt relative amounts of saturated fatty acids increased, but saturated fatty acid content of goat's milk generally decreased. Oomen (1972) described the fat distribution in Dahi. Most fat was distributed in the top layer regardless of starter addition or species of milk. As the amount of starter was increased from 1% to 2.5% the amount of fat in the top layer decreased. Stabilizers Pette and Lolkema (1951b) reported on firmness and whey separation of yogurt. Even then there was much concern about whey separation in the bottle, the commonly used yogurt package. They reported homogenization provided a beneficial effect regarding firmness. Today yogurt is packaged much differently but whey-off or phase separation is no less a problem (Kroger, 1976; Rasic and Kurmann, 1978; Richmond 3L,gl, 1982 in press). 32 Powell (1969) described use of stabilizers in cultured dairy products and reported that various stabilizers and emulsifiers aid in producing and maintaining desirable characteristics of body, texture, mouthfeel and appearance. Six groups of stabilizers are generally recognized (1) plant gums (2) manufactured gums (3) seaweed derivatives (4) gelatin (5) pectins, and (6) starches. Some stabilizers hydrate in cold water such as guar, carboxymethyl- cellulose (CMC) and certain carageenans. Locust bean gum hydrates slowly in cold water but needs to be heated (85°C) and then cooled for maximum viscosity. Gelatin will disperse in cold water but needs to be heated (60°C) and cooled before it will attain maximum viscosity. Because of these differing characteristics it may be desirable to make various blends as is often done in industry. Powell (1969) also reported that locust bean, guar and CMC disrupt the casein complex, but their reactivity is reduced by blending with carageenan and there is a certain balance between stabilizer action on casein and developing acidity in the culture. Finally, Powell (1969) noted that when used correctly stabilizers make an important contribution to cultured dairy foods. Volker (1970) discussed stabilization of fruit yogurt and noted that fruit yogurt has a strong tendency to synerese under conditions of transport and storage. Nielsen (1975) reviewed the factors that control the body and texture of yogurt. He felt that texture of yogurt was an important quality aspect and under typical handling should resist wheying-off. Factors responsible for controlling body 33 and texture of yogurt included control of (1) mix composition (2) heat treatment prior to inoculation (3) homogenization (4) starter culture and incubation conditions (5) handling ripened yogurt, and (6) stabilizer systems. Nielsen (1975) also reported that cooling, transporting and packaging were critical to protecting texture. The author further suggested that use of stabilizers is little more than patchwork. Hall (1975) described various stabilizer systems commonly used for cultured products. He reported three stabilizer systems in use were (1) gelatin (2) gelatin + plant stabilizers (3) all vegetable stabilizers. He reported swiss style yogurt, unless drinkable, required stabilization. The most widely used stabilizer was gelatin (225 or 250 bloom). Steinitz (1977) noted that the use of stabilizers is much more complex than simply increasing or decreasing viscosity. For sundae style yogurt stabilizers may be used in the upper portion but are always used in the fruit portion; and when used in both fractions they must be compatible. Recently, non-gelatin stabilizers have increased in popularity due to increasing cost of gelatin, and dietary customs (Steinitz, 1977). Stabilizers in yogurt must function in the pH range 3.8-4.5 and allow easy blending between fruit and yogurt; pectin is commonly used to stabilize fruit perserves. Steinitz reported that swiss style yogurt is often over stabilized in the U.S. A final point was that proper stabilizers in fruit and yogurt cannot resolve such problems as poor quality ingredients, improper processing, storage, transportation and handling. Oberi 25.21 (1978) studied major factors influencing consistency and stability of yogurt. 0f the factors studied 34 extending ripening time (lower temperature) proved to be the most economical and effective in increasing viscosity and reducing syneresis. Decreasing lactose content to 1.5% before fermentation prevented souring during storage but a bitter defect developed. The influence of various stabilizers on the consistency of “non heat treated stirred yogurt" with and without added fruit preparation was studied (Luczynska gL El, 1978). Commercial stabilizers were used according to manufacturers specifications. The most favorable consistency for yogurt using these commercial stabilizers was described in the article. Meiklejohn (1977) indicated Australian consumers preferred highly viscous yogurt. Traditionally viscosity is increased by stabilizers or adding extra solids or both. Many countries prohibit addition of these materials, yet concentration can be achieved legally by evaporation and membrane filtration, often complicating processing and increasing costs (Meiklejohn, 1977). He reported that with careful attention throughout the entire manufacturing scheme yogurt with high viscosity and good body and texture can be produced without the addition of stabilizers or increased solids. Samuelsson and Christiansen (1978) studied stability and viscosity of fermented milk foods. The following factors aided in the manufacture of good quality fermented milk products: milk with a high protein and fat content; storage at 5°C for 24 h before distribution; pasteurization at 85-90°C for 15 min; homogenization at 200-300 kg/cmz; moderate ripening rate; minimum mechanical treatment and proper stirring. che cul bet an; cor and all be of sam the bet 11192 5119. had $0111 (801 9601 SYSI Ofp more Ptodz 35 Using a lactodynamograph Binder (1978) studied coagulation characteristics of milk and consistency of yogurt. Eleven yogurt cultures were evaluated. There was a significant correlation between maximum amplitude and quotient of consistency; milk with an amplitude above 70 mm yielded a firm yogurt whose quotient of consistency was less than 1.0. No correlation existed between pH and consistency. Binder (1978) reported that by using a standard culture the measure of maximum amplitude with the lactodynamograph allows a judgement to be made regarding the consistency of yogurt to be expected in milk of different origin and treatment. The reaction of different cultures could then be tested using standard milk samples. Richter and Hartman (1977) used penetrometry to evaluate the body and texture of yogurt. Results indicated no relationship between body and texture ratings and penetrometer values. Penetrometer values ranged from 24.0 - 18.2. Flavored yogurts had higher values, probably due to fruit addition. Additions of 6-8% sugar also increased penetrometer values. Samples rated excellent had values of 26.2, while weak sample values were 28.9. Weak and soupy was by far the most common criticism. In summary they reported body and texture were two distinct measurements and penetrometry could not be used to rate these properties. Andres and Hagan (1977) described a new line of stability systems for yogurt. These systems were deve10ped with the objective of providing yogurt and yogurt products with longer shelf life for more efficient long distance distribution, as well as improving product texture, coSt and appearance. These stabilizer systems were 36 reported to have the ability to improve stucture and texture and possibly permit use of less solids in the mix. Some advantages for plain yogurt included low level of use (0.1%), no retardation of acid development, and improved shock resistance during transportation and storage. Nash (1980) described the use of a natural yogurt stabilizer containing only milk-derived ingredients that did not contain added stabilizers or emulsifiers. This stabilizer system reportedly produces a firm-bodied smooth textured product without phase separation. Recommended use level for lowfat yogurt was 4%. Kosikowska EL 21 (1978) evaluated 305 strains of slime-forming yogurt bacteria for their ability to improve the consistency of stirred yogurt. Eleven strains of S, thermophilus and 12 strains of L, bulgaricus were selected and 100 combinations made for further study. Criteria for selection was highest viscosity and best sensory properties. Hamdy 25.21 (1972) studied the stability of zabadi made from whole buffalo or whole cow milk, reconstituted skim and toned milk. They reported that gelatin, agar and calcium chloride improved the texture of zabadi from reconstituted milk. The use of rennet as a strengthening agent resulted in a rubbery texture with a sweet curd. Heat Treatment Heat treatment of yogurt may refer to different goals, including initial pasteurization to destroy pathogens, additional heating to 37 denature whey proteins for product stability, or to post-pasteurization heat treatment to extend product shelf life. This latter treatment has recently been the subject of much controversy, as noted earlier. Gavin (1966) studied the effects of post—heat treatment on keeping quality of yogurt at room temperature. Heat treatments depended on temperature, time and acidity; the lower the pH the lower the temperature and less time needed to increase shelf life. Shalichev and Nakashev (1973) studied the influence of pH on the equilibrium of soluble and colloidal calcium in raw and heat treated milk and yogurt. In yogurt milk (S, thermophilus, L, bulgaricus), soluble calcium increased from 33 mg/100 g at pH 6.45 to 72 mg/100 g at pH 3.70. Martinez-Castro and Olano (1980) studied the isomerization of lactose during heating (120°C). The maximum anount of isomeric sugars of lactose was 0.53 g lactulose and 0.08 g epilactose /100 9 milk. Davies 25.21 (1978) used electron microscopy to study the development of yogurt gels. They observed that protein micelles of milk heated to 95°C for 10 min possessed superficial appendages that were not apparent on protein micelles of unheated milks. They suggested during severe heat treatment denatured whey protein associated with the surface of casein micelles forming these appendages and that sulfhydryl bonding was involved in their formation. Rakshy (1966) reported that pasteurizing yogurt for a few minutes at 60°C caused high kill rates to microorganisms present and sour foods (yogurt) could be pasteurized at lower temperatures 38 .Amump .cozso. mmouoga quwmopoczowu 05H :0 VIII... “09:52.0 "+0 0...: “zwcm $0 wucwucmnwo .m 0039: 20....(mm0Eumm P3012; wIPZOE ._<¢ Dom .P< mxwm; w . v Uom .—.< wxwmg Q . N .m>mm no maid—Julm .xOmnE< NEAR—aura .xOmn—n.< mm....u..m:m .. / \ ZO_._.Jm_mm0n= 023...“. 023...". 023...". 0_Emm< 4<20Fzm>zoo ..zoo 005.05 2. _ 052505.50: pzmsimmbzuz _ _ v3.5. DmmDhJDU 39 for shorter times. Heating to 50-55°C for 30 min increased keeping quality considerably; 95 to 99% of the initial flora was destroyed under these conditions. Woods (1976) reviewed methods of higher heat, shorter time (HHST) processing and noted the terms HHST and UHT are used interchangeably. For yogurt HHST improved flavor and texture. Puhan (1979) reviewed post-pasteurization heat treatment (PPHT) of cultured dairy foods (Figure 3). Generally, a temperature of 70°C for 30-60 seconds was sufficient to eliminate LAB and contaminating yeast and molds. In yogurt 97.5% of the LAB survived a PPHT of 65°C for 22 seconds (pH 4.55) whereas 99.9% of the LAB were eliminated at this same time/temperature combination (pH 3.82). He reported the purpose of heat treating cultured dairy products was to prolong shelf life while maintaining product quality. Speck and Geoffrion (1980) reported PPHT inactivates starter cultures as well as also inactivating ES-galactosidase. Heat was more damaging to lactase at pH 4.2 than pH 4.6. No lactase activity or viable culture bacteria were detected after heating at 70°C for 2 min. Yogurt Cultures The lactic acid bacteria (LAB) include species of the family Lactobacillaceae. They are structurally a heterogeneous group but are characterized by their main end product--lactic acid. They are gram positive, non-sporulating rods or cocci. Their catalase 40 activities vary. These bacteria are widely used by the dairy industry throughout the world. Various authors have reviewed the role of LAB in cultured dairy products (yogurt) manufacture (Speck, 1979; Sharpe, 1979; Moon and Reinbold, 1974; Vedamuthu, 1974; Gordon and Shapton, 1977; Tramer, 1973). Tramer (1973) described qualities that good yogurt starters should have (Table 3). Gordon and Shapton (1977) discussed general characteristics of yogurt starters. Yogurt cultures should be obtained from reputable commercial culture houses or from research institutes. Cultures should be renewed at regular intervals (Tramer, 1973). Handling cultures requires certain skills as well as awareness of different temperature optima, differing growth rates, symbiotic or associative growth characteristics, and importance of maintaining culture balance (Tramer, 1973). Many aspects of culture maintenance were discussed by Tramer. In Figure 4, the inhibitory effect of various sugars on acid development is depicted. These results reveal a marked inhibition of cultures with increasing sugar content. Microscopic examination revealed the rods were the organisms affected. However, Tramer concluded that increased sugar concentration was not the problem but rather increased total solids (T.S.) was the critical factor. Above 22% T.S. severe inhibition occurred and further noted cultures varied in their resistance to increased T.S. The selection of a suitable starter is therefore not an easy matter. Speck (1979) reported that little work has been done on starter cultures used in yogurt manufacture. The primary purpose of cultures in yogurt manufacture are for the production of flavor compounds, lactic acid, and texture, which result from starter growth and acid production (Speck, 1979). 41 Various combinations of L, bulgaricus and S. thermophilus were used to define acid and flavor production and proteolytic activity in skim milk medium (Singh and Sharma, 1982). Variations in titratable acidity, volatile acidity, acetaldehyde and amounts of liberated tyrosine were noted. However, one of the combinations (Lb-RTS, St-HST) showed much higher acidities and more acetaldehyde production compared to other starters. Results agree with other authors regarding the higher proteolytic activity of L, bulgaricus. Moon and Reinbold (1974) discussed selection of active and compatable starters for yogurt. They reported yogurt flavor depended on the acetaldehyde-lactic acid ratio. The compatibility of cultures used in yogurt manufacture was an important criterion for strain selection. These authors described a procedure (modified coagulase test) for proper strain selection of yogurt cultures. 252 possible combinations were studied (21 S. thermophilus, 12 L, bulgaricus) and 33 controls. Under test conditions, pairs that required longer times to coagulate than controls were considered inhibitory; pairs that required the same time as controls, intermediate; pairs that required shorter time than controls, stimulatory. Coagulation time differences between inhibitory and stimulatory pairs was as much as 2 h. This type of testing could be important to industry since microbial interactions can affect the overall character of yogurt. No review about yogurt would be complete without mention of some of the important early research (Pette and Lolkema 1950 a,b,c). Pette and Lolkema (1950a) described acid production and aroma 42 formation in yogurt. They reported typical yogurt aroma was due to (l) lactic acid, and (2) aroma substances produced; the aroma compounds were produced by the rods which developed as acidity increased. They identified acetaldehyde in the distillate. In a second article (Pette and Lolkema, 1950b), they found yeast autolysate lengthened log growth phase and stimulated acid production in S, thermophilus. They reported S. thermophilus required certain amino acids which were not present in sufficient concentration in the original milk. In mixed culture, L, bulgaricus hydrolyzed portions of milk proteins thereby stimulating acid production by the cocci. The most important amino acid liberated was valine, and concentration of this amino acid in milk varied during the year. In the third article Pette and Lolkema (19500) showed that mixed cultures produced more acid than single cultures. Rapid acid production was due to stimulation of the cocci by the rods. In addition to this symbiotic action, they also demonstrated an inhibitory effect of rods on the cocci due to lactic acid production. Galesloot 25.21 (1968) reported that S, thermophilus stimulated L, bulgaricus by producing a factor that was equal to or replaced by formic acid. However, this stimulation could only be demonstrated in moderately heated milks. These authors reported that other researchers were not able to observe the stimulation of L, bulgaricus by S. thermophilus because they used steamed or sterilized milk containing formic acid formed by the severe heat treatment. Shankar and Davies (1977) briefly reviewed associative 43 bacterial growth of yogurt starters. They reported some compound(s) other than an amino acid was the major stimulatory component produced by L, bulgaricus, possibly certain peptides derived from milk casein. Singh and Ranganathan (1979) discussed the caseinolytic activity of L, bulgaricus and a mutant produced using N-methyl-N-nitro-N-Nitrosoguanidine. The mutant released greater amounts of tyrosine when compared to the parent culture. Both the mutant and parent cultures degraded k-casein more readily than as or 13-fractions. Sharpe (1979) reviewed culture symbiosis, noting that L, bulgaricus has a more active cell bound proteinase than 'S. thermophilus which aids the growth of S, thermophilus by releasing stimulatory peptides or amino acids from casein. McKay 23.21 (1971) reviewed the biochemical nature of lactose utilization by LAB noting lactic acid contributes to flavor, color, texture and keeping quality of cultured dairy products. Lactose hydrolysis by S. Sgrgug and lactic streptococci was discussed. Lactose can by hydrolyzed and transglycosylated by the enzyme 8 -galactosidase. The mechanism of enzymatic synthesis of galactosyl oligosaccharides was described by Pazur (1953). He described the synthesis of four galactosyl oligosaccharides during the hydrolysis of lactose by a yeast enzyme. Asp 3L El (1980) also discussed the structures of various oligosaccharides produced during the hydrolysis of lactose. Rutter and Hansen (1952) described the conversion of galactose to glucose derivatives in L, bulgaricus. Carbohydrate or lactose content of yogurt varies widely among commercial yogurts. Goodenough and Kleyn (1976a) reported lactose 44 501103 1111.01 % .Amum. .0000... 0020.30 00:00» a mo u:00¢o.0>0u 0.0a co m:0...uue 000nm mso.sm> mo 000000 x000.0.;=. .0 0030—; 0.9.: 0.50... 2. ms...- 0 o m a F 0 _ . _ _ _ o. Iluoool «.|dl|\ III II. ‘0... \000‘. o \o \\ 0.31:1...200053 .. \ “\ Ind ..\0 \ .Azme? \ .. \ozo .\\ oéal .A0 \0 1.0.. ..\\\wam. .\\nv \$A@ \0@ \ O o 0 III. \ 09‘ m E \ 1 m; \ . 0.09 I h p ('V"l %) MIOIOV mauve“; 45 content of fresh yogurt (8.5% lactose) decreased to 5.75% after fermentation. Glucose remained at trace levels throughout incubation and galactose increased from trace to 1.2%. Lactose content in commercial samples ranged from 3.31 - 4.74% and galactose from l.48 - 2.50%. Because of the amounts of lactose found, these authors questioned the often-made assumption that lactase deficient individuals can tolerate cultured milk products better than non fermented products. As mentioned previously, Goodenough and Kleyn (l976) reported laboratory rats fed natural yogurt containing viable cultures were able to absorb galactose more efficiently and intestinal lactase activity was greater. Culture survival was demonstrated in 2112 up to 3 h after feeding. Kilara and Shahani (1976) discussed B-galactosidase activity of cultured dairy products including yogurt and a direct acidified yogurt product. Cultured yogurt possessed considerable enzyme activity, while the direct acidification product showed no enzymatic activity. Because LAB are nutritionally fastidious, one must expect some change in vitamin content during incubation of yogurt. Acott and Labuza (l972) and Reddy gt 31 (l976) reported on vitamin content of ripened yogurt. Reddy gt 31 (1976) compared the content of cultured and acidified yogurt after ripening and storage. Folic acid and 812 content decreased 29 and 60% in cultured yogurt and 48 and 54% in acidified yogurt stored at 5°C for 16 days. Biotin, niacin and pantothenic acid remained relatively stable throughout storage. Deeth and Tamime (1980) and Rasic and Kurmann (1978) provided an in-depth review of vitamin content of yogurt. Okonkwo and Kinsella 46 (1969) found the content of orotic acid decreased from 8.2 - 4.6 mg/100 ml during ripening of yogurt. This was attributed to action of the lactobacilli. Upon cooling, orotic acid levels remained constant and yogurt contained about one-half the concentration normally found in milk. Culture Enumeration Sandine 25 31 (1976) reported on acid producing organisms and, in particular, LAB in the Compendium of Methods for the Microbiological Examination of Foods. Current methods as well as 'reagents and necessary media were described. Rasic and Kurmann (1978), Tamime and Deeth (1980), and Tramer (1973) also provided information regarding microbiological assessment of LAB. In 1971 Davis gt al_reported on enumeration and viability of L, bulgaricus and 5. thenmophilus in yogurt. At that time few methods had been described for differentiating and enumerating yogurt bacteria. Using a double pour plate technique with media containing mildly reducing substances, a satisfactory method was found that differentiated yogurt bacteria by colony type under the microscope. The medium provided easy and reliable differentiation -- §. thermophilus being smooth, round or lenticular, and L. bulgaricus showing rough or irregular shaped colonies in the depth of the medium (Davis 32 31, 1971; Tramer, 1973). Another medium for the differential enumeration of yogurt bacteria was described (Lee 23 _al, 1974). They reported that all §, thermophilus strains fermented lactose and sucrose, while L, bulgaricus fermented lactose but not sucrose. Commercial yogurts were successfully tested for 47 differential counts on Lee's agar. §, thermophilus produced yellow colonies, while L, bulgaricus appeared as white colonies (less acid production). For enumerating and separating organisms in yogurt culture, Shankar and Davies (1977) used the inhibitory property of B-gly- cerolphosphate toward L. bulgaricus to selectively isolate and enumerate §. thermophilus. Johns gL'aL (1978) used reinforced clostridial agar (RCA) to suppress the growth of §, thermophilus thereby allowing selective isolation 0f.L- bulgaricus. Both Shankar and Davies (1977) and Johns gL 31 (1978) used the LAB procedure (Davis 2L 31, 1971) in confirmatory work. Monk (1979) used microbial thermograms to investigate the growth of §. thermophilus and L, bulgaricus in single and mixed culture (milk medium). Thermograms are a continuous record of the rate of heat production. Monk (1979) reported the area enclosed by a thermogram is proportional to the heat produced and has three main components. Catabolism of lactose provided the largest amount of heat. He reported the heat effect will decrease when growth stops, when concentration of a substrate or a growth factor limits growth, or rate of catabolism decreases due to inhibition by metabolites, including changes in pH. In single culture, L, bulgaricus produced 48% more heat during a 9 h period than §, thermophilus. The maximum heat effect reached during growth is a measure of cell yield. Rasic and Kurmann (1978) and Hup and Stadhouders (1972) described media and enumeration of yeast and molds in yogurt. Hup and Stadhouders (1972) compared media for enumeration of yeasts and 48 molds in dairy products. They recommended for sour dairy products oxytetracycline glucose yeast extract agar (OGY). Culture Activity Kondratenko 2L 31 (1978) described the inhibitory effect of antibiotics on yogurt production. Penicillin and five other antibiotics tested revealed varying results. Penicillin and streptomycin at low concentrations (.007 units/cm3 and 0.8 3, respectively) inhibited yogurt microflora. When 3 mg/cm penicillin concentration was 0.2 units/cm there was no growth. Chloramphenicol had the least inhibitory effect. Rubin (1976) studied factor(s) in yogurt responsible for inhibiting the milk borne pathogen g, typhimurium. Rubin reported although there has been much work in this area, specific factors responsible for the death of this organism have not been clearly identified. Lactic acid was reported to be chiefly responsible for inhibiting this bacterium. A direct correlation was observed between intracellular dissociated lactic acid species and inhibition of growth rate of_§. typhimurium, but no correlation was apparent for the intracellular undissociated moiety. Cousin and Marth (1977) manufactured yogurt from skim milk precultured with psychrotropic bacteria. Yogurts made from milk supporting growth of normal flora and Pseudomonas 33. (No. 36) were not statistically different from control yogurt. Yogurt made from milk precultured with Pseudomonas 52. (No. 13) and Flavobacterium s3, (No. 26) were unacceptable. Meikljohn (1978) described a seasonal inhibition of yogurt 49 cultures in Australia. He reported that a combination of seasonal conditions and particular manufacturing conditions used to prepare the fruited yogurt possibly lead to periodic culture inhibition. Microscopic examination revealed normal cocci but rods were nearly absent. Inhibition was overcome by reducing total solids to 22%. He suggested relatively high osmotic pressures predisposed the lactobacilli to an inhibitor that becomes apparent during a certain period of the year in heat-concentrated non-fat milk. However, this inhibitory phenomenon was transient, persisting only a few weeks. Peake and Stanley (1978) described the specific inhibition of L, bulgaricus during commercial production of yogurt. A scheme was described to determine presence of the bacteriophage. Electronphotomicrographs revealed phage possessed hexagonal heads of 60 nm diameter and tails of 210 nm length. Mikolajcik and Hamdan (1975a) discussed growth characteristics and metabolic by-products of L, acidophilus. Experiments using skim milk medium were conducted to determine growth rates, optimum temperature for growth, and viability upon storage. Generation times ranged from 49 min to 104 min. Storage at 5°C resulted in little loss of viable cells. In another article these authors reported an antibiotic isolated from L, acidophilus (acidolin). After purification acidolin exhibited the following properties: (1) dialyzable with low molecular weight ca 200 (2) no nitrogen (3) highly hygroscopic (4) very acidic (5) active against a wide range of organisms, including spore formers but not LAB (6) very heat stable (121°F, 15 min), and (7) non-toxic to tissue culture cells. 50 Shahani gLHgL (1976) investigated the antibiotic production potential of various strains of L, acidophilus and L, bulgaricus. Milk was essential for production of these antibiotic substances. In another article Shahani 3L.gl (1977) detailed the procedure for isolating acidophilin. Purification fold was 247 with a 4.5% yield. The amount of acidophilin required to cause 50% inhibition of 27 bacteria was determined (17 were common pathogens). In general 30-60 Eg/ml acidophilin was required for 50% inhibition, ascribing some possible therapeutic properties to acidophilus milk (Shahani gL‘gl, 1977). Speck (1975) reported literature has generally indicated that intestinal well-being is associated with high numbers of fecal lactobacilli. He reported when fecal samples are plated with lactobacillus selective agar and incubated in an atmosphere of C02, the microaerophilic lactobacilli, including L, acidophilus, can be enumerated. Speck (1975) also described developments in acidophilus products. Bauer 23.21 (1976) reported that fresh milk contained more bound than free acids. During the souring process bound, free, and total acids increased progressively through 90 min incubation and then rapidly thereafter. Free acids increased the most (208%), and bound acids the least (82%). This distribution was reported to be in correct relation to T.A. and pH values. Steffen 3L.gl (1973) studied configuration of lactic acid formed by different lactic acid bacteria in relation to processing conditions. All §. thermophilus strains tested produced over 92% L (+) lactic acid, while L, bulgaricus strains produced 0 (-) lactic 51 acid. Puhan 2L a1 (1973) selectively isolated and enumerated lactobacilli (Rogosa agar) and streptococci (Streptoselagar) in 269 yogurt samples. Average bacteria counts were 500 million/ml with streptococci making up 60-80% depending on age and pH of the yogurt. 0 (-) lactic acid content increased during storage as a result of metabolic activity of the lactobacilli, whereas L(+) lactic acid remained constant throughout storage. Enumeration of §, thermophilus and L, bulgaricus on selective media revealed an increase of streptococci in the early phase of incubation (Puhan EL ‘21 1973b). The acidity of the yogurt changed in correlation to the activity of the LAB. After reaching the maximum count of LAB the content of L(+) lactic acid stabilized, whereas D(-) lactic acid increased quickly in the beginning of storage but increased more slowly throughout storage. Lacrosse (1970, 1972) discussed development of acidity in yogurts stored at varying temperatures. He concluded that yogurt should not exceed .90% lactic acid if its flavor is to be acceptable, and under proper storage yogurt could be held at 5°C for 15 days, or 10°C for 5 days (Lacrosse, 1970). Fluckiger and Halser (1973) studied the effect of initial pH on susceptibility of yogurt to souring (post-acidification) during storage. Results indicated that yogurt with higher initial pH soured more than yogurt with low pH, especially in the first 14-18d. But this was not an absolute relationship. Abrahamsen (1978) used whole milk and five different yogurt cultures to examine the content of lactic acid and acetaldehyde in yogurt stored at different temperatures. During 52 storage the proportion of L(+) lactic acid, (as a percent of the total) decreased for all samples indicating L, bulgaricus activity in cold storage. Different starters showed varying amounts of acetaldehyde production. For some samples the content did not change through 24 d storage, while for others acetaldehyde decreased considerably throughout storage. In four of five cultures examined, acetaldehyde levels dropped during storage. Flavor of Natural Yogurt Pette and Lolkema (l950a,b,c; 1951a,b) published a series of articles providing some basic knowledge about yogurt. In some of the research (Pette and Lolkema, 1950c) they reported that two primary components were responsible for yogurt flavor: (l) lactic acid, and (2) aroma substance(s) produced. For best yogurt flavor an acidity of 0.85-0.90% was desirable. They also reported that the lactobacilli were responsible for lactic acid production during storage and they identified acetaldehyde as a component of yogurt flavor. In another article (Pette and Lolkema, 1951a) the optimum proportion of rods to cocci was determined for proper and typical yogurt flavor. The optimum ratio was 1:1, a practice which is now commonly accepted in commercial yogurt fermentation. Harvey (1960) quantitated acetaldehyde and acetone production of various lactic streptococci. Cultures were grown in autoclaved skim milk and the carbonyl compounds determined using PC. Acetaldehyde content ranged from 0.5 ppm to 10 ppm in the cultures. According to threshold sensory data these concentrations should impart significant effects on flavor and aroma of milk cultures (Harvey, 53 TABLE 4 Acetaldehyde content and acidity of various yogurts.a Yoghurt pH T.A. CH3CH0 L. bulgaricusb+ 4.33 1.55 34 ppm S. thermophilus L. bulgaricusc+ 4.4 1.33 25 ppm sodium formate S. thermophilusd+ 4.5 1.37 25 ppm casein hydrolysate Commercial samples 4.03 2.04 20 ppm of yoghurte 3Marshall and Mabbitt (1980). Mean of 8 samples. cMean of 6 samples. ean of 14 samples. eMean of 5 samples purchased from local supermarket. 54 1960). Acetone content was generally less than 1 ppm. Quantities of acetaldehyde and diacetyl produced in skim milk and MRS media by 5 species of streptococci were reported (Bottazzi and Dellaglio, 1967). In both media all strains of S. theanphilus formed more acetaldehyde and diacetyl than other homofermentative lactic streptococci. Cultures of all isolates of S, thermophilus contained mean concentrations for acetaldehyde and diacetyl of 3.0 and 1.0 ppm in skim milk and 3.5 and 3.0 in MRS medium, respectively. They reported single strain cultures produced acetaldehydezdiacetyl in an unfavorable ratio for balanced flavor. In a recent article (Marshall and Mabbitt, 1980) yogurt of typical flavor was produced using single starters with certain prescribed additives (Table 4). Yogurt was made successfully on a laboratory scale using single starter organisms. A 2% inoculum of .S. thenmophilus and 0.25% casein hydrolysate or a 2% inoculum of L, bulgaricus and 30 ppm sodium formate was used. Resulting yogurts had titratable acidities and acetaldehyde contents comparable to mixed culture yogurts (Table 4). The advantages and limitations of using single starter organisms for commercial yogurt production were discussed (Marshall and Mabbitt, 1980). Yogurt made using only S. thermophilus does not exhibit over acidification during storage and sugar content can be increased more in the presence of the cocci. Bottazzi and Vescovo (1969) reported on quantities of carbonyl compounds produced in skim milk medium from 84 strains of thermophilic lactobacilli isolated from commercial yogurts. Many strains were very active producing 15-20 ppm acetaldehyde; however, 55 many strains were also relatively inactive. They reported cultures producing about 8 ppm acetaldehyde gave rise to good flavored yogurt, whereas cultures producing less than 4 ppm did not have full flavor. Of all thermophilic lactobacilli studied, none produced diacetyl, and only small amounts of acetoin were formed. Cultures producing an acetaldehyde:acetone ratio of 2.8:1 had stronger yogurt flavor in milk. Acetaldehyde production of different commercial starters was determined through 7 h incubation at 45°C, as well as through four weeks cold storage at 4°C (Hamdan engL, 1971). Maximum acetaldehyde (23-27 ppm) was produced by the fifth hour of incubation. During storage some cultures reduced acetaldehyde and some did not. When single strains (of each organism) were used, acetaldehyde content was reduced considerably compared to acetaldehyde production of combined commercial cultures. Although L, bulgaricus produced more acetaldehyde than S, thermophilus, combining cultures (1:1) resulted in increased production of acetaldehyde (Figure 5). In an interesting paper (Collins, 1972) biosynthesis of flavor compounds produced by lactic streptococci and associated organisms was reviewed. Collins reported diacetyl, acetoin and 2,3- butylene-glycol were closely related compounds representing three levels of oxidation of one four-carbon skeleton. He reported that the majority of microorganisms that synthesize diacetyl are also able to reduce it to acetoin; acetoin may then be further reduced to 2,3 -butyleneglycol. Biosynthesis of acetoin, acetyl-coenzyme A and diacetyl were discussed. Collins also discussed the importance of 56 these reactions and why microorganisms produce such metabolites. Influence of pH and aeration on their formation was also described. Dumont and Adda (1978) reviewed flavor formation in dairy products. Flavor compounds resulting from carbohydrate, lipid and proteins were described. They reported that during ripening amines are formed through amino acid decarboxylation, and while their flavor significance is still questionable, biological amines are of concern because of possible health hazzards to certain susceptible individuals. Groux (1973) studied yogurt flavor. He reported when acetaldehyde was present in small quantities diacetyl and even acetoin could partially substitute still providing typical yogurt flavor. Groux further suggested that free amino acids resulting from proteolysis could be precursors of some aromatic compounds contributing to the overall flavor of yogurt. Gorner 23.21 (1975) studied changes of volatile substances in yogurt during ripening with the aid of GC. Acetaldehyde content of ripened yogurt ranged from 23.1 to 33.0 ppm. They reported acetone, 2-butanone and ethanol were all present in milk used for yogurt production. Gorner 3L El (1978) studied the volatile compounds in yogurt (21% TS) using gas liquid chromatography (GLC). Satisfactory yogurt flavor I coincided with acetaldehyde contents ranging from 23-47 ppm. There have been many different values reported for the levels of compounds needed to produce typical yogurt flavor. There is much yet to be done to adequately describe the flavor of natural yogurt, however, Groux (1976) studied components of yogurt aroma. Using 57 .23.; .1... Mm .522: 5m. 0m 0.:0st pup a 0:0 030,00 pan.,...mappgaoEngu .m no :.0000 0pmcvm 0 aa.:ovuuauoga 0vag0u_0u00< .m 0gampm 601.02.... . m s. o m e . m a w _ . _ . _. . _ u 4\"\ O o o o o 0.20 n. 002.020wollo 03.03.50 ...o|lo 03.13.2005 .01 CV F co EOAHBCI'IVLSOV de CD CM Sf CV 00 CV 58 freeze dried cultures cultivated in autoclaved skim milk containing 1% yeast extract (YE), volatile components (acetaldehyde, diacetyl, acetoin) and non-volatile components (free amino acids) were quantified and related to yogurt flavor. Bacterial strains used did not show any proteolytic specificity since all amino acids, except arginine and cystine appeared in free form and quantities of free amino acids were not in ratio to their concentration in milk proteins (Groux, 1976). Generally free amino acid content increased during ripening, but glycine and lysine were consumed by the culture. Ammonia nitrogen increased during ripening and proline was the most abundant amino acid produced during fermentation. In summary, Broux reported that free amino acids did not contribute much to yogurt flavor but they might be precursors of important volatile compounds. He also concluded from trained panel sensory data that diacetyl and acetoin were important contributors to typical yogurt flavor. Viani and Horman (1976) identified numerous compounds present in yogurt aroma. They reported the mild technique used in the isolation of the aroma complex allowed them to distinguish between compounds resulting from the heat treatments used during the preparation of yogurt and eventual laboratory artifacts. Numerous aroma compounds formed during manufacture as well as possible precursors are shown in Table 5. Using GC headspace analysis, Hild (1979) quantitated acetaldehyde, ethanol, acetone and diacetyl in yogurt. He noted yogurts with distinct flavor contained at least 5-7 ppm acetaldehyde, and yeasty and spoiled products were 59 characterized by increased ethanol contents. Acetaldehyde content of these spoiled products was also increased. Brandao (1980) determined volatile flavor components of yogurt using a headspace GC technique. He reported acetaldehyde production of 25.5 ppm for mixed culture (1:1); ethanol and diacetyl content increased during fermentation. Brandao discussed the importance of ethanol dehydrogenase activity. During storage of yogurt (after 14 d) ethanol increased concomittantly with decreasing acetaldehyde content. Hamdan eL_al (1971) describing the effect of potassium sorbate on cultures found that potassium sorbate (0.05%, 0.10%) retarded growth of both yogurt bacteria as well as decreasing acid production of cultures incubated at 45°C. Acetaldehyde in these cultures decreased during storage but decreased more so in samples containing potassium sorbate. Various authors (Gorner.eLiaL, 1971; El-Sadek 2L.al, 1972; Abrahansen 3L _a_l, 1978; Singh 35 a_l, 1980) have compared volatiles formed during manufacture of yogurt using milk from different species of mammals. Gorner 3L El (1971) found that acetaldehyde concentration was greatest in yogurt made from cows milk, followed by goats milk and finally sheep milk. Abrahamsen 2L.al (1978) compared the growth of yogurt bacteria, acid development and volatiles formation in cow and goat milk yogurt. Acetaldehyde content was lower in goat milk yogurt. Cows' milk yogurt contained 17 ppm acetaldehyde at 3 h but decreased to 13 ppm after 8 h, whereas goat milk yogurt contained 5 ppm after 3 h incubation and increased to 9 ppm after 8 h incubation. Goats' milk yogurt 60 TABLE 5 Compounds in yogurt aromaa. Constituents of microbial origin CzH40 Acetaldehyde From the C4H502 Diacetyl lactose-citrate C4H302 Acetoin cycle C5H302 Acetylpropionyl Either from C5H1002 2-Hydroxy-3-pentanone the threonine 3-Hydroxy-2-pentanone cycle, or thermally from fats Constituents formed by thermal degradation of fat. C3H60 Acetone From Ketoacids (4) C4H80 Butanone Cngo 3-Pentene-2-one C5H120 .2-Hexanone C7H140 2-Heptanone CgH130 2-Nonanone 011H220 2-Undecanone C5H802 Y'-Valerolactone From hydroxyacids (4) 05H1002 00 00000000.“ 00020 ...0.0_.0.0 00...0_00.00 00...“ 00.0 00.0.0.00.0 mmmmaem “W0 . I . . I . O I . . 0 III . AQF " zv 0. 0 + 00 0 00 . + 00 00 00 . + .0 0 0. 0 + .0 0 0.0000 0.0 ..00 e 1 e e I e e ll e e 1.. e AQN H 2v 0. 0 + .0 0 0. 0 + 00 00 00 0 + 00 . . 00 0 + 00 0 0.000. 000 :0. =0 .0. .0. .0. 0.2003 00..00 .000. 00. 0.0000. 00000.0 0.000000» 00go>0pm 0:0000> mo 00.0.000500 .0005000 .0 0.00» .copu0p>0o 0000:000.H_:0020 00.0.0 00.0 .0 u z. 00.0.0.00.0 00.. + 00.00 0..0.u 00.0 0 000.0 e I e e I e e I e e .1 .e AD "2V .. 0 + .0 0 00 0 + 00 .0 00 0 + .0 . 00 0 + 0. e >. 000.0 s I e e III e e I e. e I e AMP "2v .. 0 + 00 0 0. . + 00 00 0. 0 + 00 . 00 0 + .0 0 .. 000.0 ...0.0 0..0 0..0.H 00.00 00.0.0 00.. 00.0.0 00.0 .w meuw0 :01. .0. An. .0. zeom0000 000.00 .000. 0.. 0.000.. 0000000 0.0gamo> c0go>0P0 0001300 mo mu=0gm 0:o_gm> mo 00000000500 .0005000 .. 0.000 111 Handbook 8-1 (10). However, Handbook 8-1 gives values for all types of yogurt except full-fat fruit flavored yogurt.‘ Therefore, no general com- parisons can be made for full-fat flavored yogurts. It would seem advisable, with increasing production and consumption of yogurt in the U.S., to make available in the future published data on full-fat flavored yogurt. In comparing the data in Table 2, one finds that in addition to the higher fat content, protein and total solids content were also found to be greater in the full-fat brands analyzed. The pH values were compara- tively different; the mean pH of full-fat products was 3.88 compared to an average of 4.07 for the low-fat yogurt. Composition of Plain Yogurt Both full- and low-fat plain yogurts surveyed (Table 3) were similar in protein content. Mean values for percent fat, percent total solids and pH were all found to be greater in the full-fat yogurts. In comparing the data for low-fat flavored and low-fat plain (Table 2 vs. Table 3) the results indicated higher pH, protein and fat content in the low-fat plain yogurt while the flavored low-fat yogurts had larger values for percent total solids. The results were similar for full-fat yogurts (flavored vs. plain) in that pH, protein and fat content were all found to be greater in the plain full-fat yogurt samples analyzed. Mean total solids contents were greater in the full-fat flavored yogurts as would be expected, and mean pH values for full-fat flavored yogurts were notably less than for full-fat plain yogurt. 112 .00.00.>00 0.000000.“ 000:0 e I e e 1 e e l e e I e Am " 2V 00 0 + 00 0 00 . + 00 0. 0. . + 00 0 00 0 + 00 0 . 00.0000 ..0 00.0.0 00.0 .0.0.0 00... 00...0 ...0 00.0.0 00.0 0.000. Mm.u.aw. ...0.0 00.0 .0...0 00.0. 00.0.0 00.. 0..0.0 00.0 0.000..M0H “w. :0 .0. E .00 0.000000 00..00 .000. 000 0.000.. 00000.. 0.0.000. 0.0.0 mo 0000.0 0:0..0> we 00.0.0oasou .00.E0=u .m 0.00. 113 Net Heights of Flavored Low-Fat Yogurt Net weights are shown in Table 4. Overweight seemed to be a common denominator of low-fat flavored yogurt (actually of most yogurt examined) with a mean value for the 28 yogurts surveyed being nearly 5% overweight. Brand I had a mean net product overweight of about 2.2%. Brand II was a striking 7.5% overweight, while Brands IV and V had a net product over- weights of 2.3 and 1.3 respectively. Considering all low-fat flavored yogurts surveyed, results showed a range from l.5% under declared con- tainer net weight to as high as l2.6% over declared container net weight. In summarizing the data for full-fat yogurts (not shown), Brand IV on the average was 4.7% greater than its declared container net weight, while Brand III averaged only 0.4% overweight. In general, it appears from these data that yogurt consumers are getting more than they are paying for. Caloric Content of Yogurt Table 5 shows estimates of caloric content, cal/loo g, of various commercial yogurts. These values are based on the caloric equation used by Kroger and Weaver (7). The mean caloric values for flavored low-fat and full-fat yogurts were l06 and lZl cal/100 g, respectively. Mean values for plain yogurt ranged from 69 cal for low-fat plain to 92 cal/lOOg for full-fat plain. From these data it is apparent that low-fat plain yogurt contains 25% less calories than full-fat plain yogurt. Furthermore, it is evident that in relation to low-fat plain yogurt, full-fat and flavored yogurts account for a much increased caloric density on a per container bases. Flavoring addition alone accounted for approximately 30 cal/loo g for both the full and low-fat samples surveyed. Also of interest are the caloric ranges found in flavored yogurts. Low-fat 114 Table 4. Net weight of various brands of flavored low-fat commercial yogurts.a Net Neightb Product Category (9) Brand I 231.9 :_3.69 Brand II 244.0 i_5.15 Brand IV 232.6 :_2.63 Brand V 229.9 :_5.83 All Samples (N = 28) 237.8 :_7.78 aMean : standard deviation. bDeclared weight = 227.0 9. Table 5. Calculated Caloric Content of Various Commercial Yogurts.a Product Category Calories/100 g Flavored: . Low fat (N = 28) 106 :_ 9 Full fat (N =13) 121 :10 Plain: _ Low fat (N = 3) .69 :_ 4 Full fat (N = 2) 92 :_20 aMean : standard deviation. 115 flavored yogurt ranged from 80 - 120 cal/100 9 while full-fat flavored samples ranged from 102 - 135 cal/100 9. Considering the wide variation in caloric content of market yogurts along with an overfill of almost 5% for all flavored samples surveyed, the caloric content of many yogurts may be substantially greater than the value indicated on the container. The data presented indicate that there is still much variation in yogurt composition not only between brands but within the same brand. Moreover, the results of the analyses obtained in this survey are in general agreement with those reported in the literature (5, 6, 7) in that wide variations were observed in gross composition. Apparently there has been little effort to standardize yogurt during the past 7 years despite the fact that better uniformity in composition and quality would be bene- ficial to both consumer and producer. REFERENCES Anon. 1978. Milk facts. Milk Industry Foundation, Washington, D.C. Anonymous. '1978. Yogurt sparks market as dairies play it safe. Dairy Ice Cream Field 161 (7):42-43. Association of Official Analytical Chemists. 1975. Official methods of analysis. 12th ed. AOAC, Washington, D.C. Chandan, R.C. _1977. Considerations in the manufacture of frozen and soft serve yogurt. Food Prod. Dev. ll(7):118, 119, 121. Davis, J.G. and T. McLachlan. 1974. Yogurt in the United Kingdom: Chemical and microbiological analysis. Dairy Indus. 39(5):l49, 150, 152, 154, 157, 177. ' Duitschaever, C.L., D.R. Arnott, and D.H. Bullock. 1972. Quality evaluation of yogurt produced commercially in Ontario. J. Milk Food Technol. 35:173-175. Kroger, M., and J.C. Weaver. 1973. Confusion about yogurt - compositional and otherwise. J. Milk Food Technol. 35:388-391. Mojonnier, T., and H.C. Troy. 1927. The technical control of dairy products. 2nd ed. Mojonnier Bros. Co., Chicago, IL. Quackenbush, 0.6. 1978. More Aermicans liking yogurt more and more. Dairy Record, 79(2):47-50. United States Department of Agriculture. 1976. Consumption of foods; dairy and egg products, raw, processed, prepared. Agriculture Handbook No. 8-1. Gov't Printing Office, Washington, D.C. 116 CHAPTER III PRODUCTION, PROCESSING AND SENSORY EVALUATION OF SWISS-STYLE HONEY YOGURT] 1To be submitted to J. Food Protection. 117 INTRODUCTION The great popularity of yogurt in the U.S. has been attributed by some to the diversity of fruit, fruit preserves or other flavorings that have been introduced in the yogurt base. Currently, about 50 flavor bases are available for addition to plain (natural) yogurt. Fruit is added to yogurt in various ways: (1) Swiss - fruit and yogurt are com- pletely mixed yielding a homogeneous product, commonly stabilized, (2) Sundae - fruit is first metered into the container and yogurt base is added prior to culturing in the container. The ”natural food“ trend and increased advertising expenditures have also aided market growth of yogurt. Various researchers (Brown and Kosikowski, 1970; Duthie gt_al,, 1977b) have incorporated flavorings such as honey and maple syrup into yogurt with varying results. Brown and' Kosikowski (1970) found that darker buckwheat honey was preferred over a light colored clover honey. Duthie gt_gl, (1977a, b) manufactured varying swiss-style maple syrup yogurts (8 - 18%), developed a score card for evaluation and then performed sensory evaluation to define the pre- ferred syrup levels. Because many people consider yogurt and honey as natural foods, and because lower honey levels may be used, providing desired sweetness and reduced caloric density low fat honey yogurt may prove to be an appro- priate food either as yogurt or as yogurt base whereby additional fruit or fruit preserve may be incorporated. Another concern is stabilizer addition. Many experts and lay people belive yogurt need not have added stabilizers to produce a good bodied yogurt. Because of tradition as well 118 119 as increasing costs yogurt is often made in the home and commercial yogurts serve as starter culture. Home produced yogurts rarely contain added stabilizers or emulsifiers. Using buckwheat honey, skim milk solids, whole milk, water, and yogurt bacteria isolated from commercial yogurt we manufactured and evaluated swiss-style low fat honey yogurt without stabilizer addition. Various honey levels as well as honey addition before and after heat treatment were evaluated by experienced dairy prOduct judges as well as by a larger untrained consumer population for various sensory attributes. MATERIALS AND METHODS Yogurt Processing, Whole milk was obtained from the Michigan State University Holstein dairy herd. Buckwheat honey was purchased locally. Plain yogurt mix was standardized to 1.5% fat, 12.6% solids non fat with the addition of skim milk solids and water (14.1% T5). The amount of honey used ranged from O - 15% by weight. These additions are more fully discussed later in the paper. TM mixer Ingredients were blended together with the aid of a Lightnin and pasteurized at 88°C, 40 min. The mix was then cooled to 60°C and homogenized at 70.3 kg/cm2 first stage and 35.2 kg/cm2 second stage. The mix was cooled to 43°C and a previously isolated commercial culture added (no slime forming capability) to ripen the yogurt. Honey was either added before pasteurization (8P) or at the time of inoculation (AP). Yogurt was ripened at 42 - 43°C until pH 4.5 and then stored at 5° for sensory evaluation either by experienced dairy product judges or by con- sumer panelists. For consumer evaluation, yogurt was carefully transported by car to the University of Guelph, Ontario where expert personnel aided in experimental design for statistical analyses and scoring procedures for evaluation of 5% and 6% honry yogurt. Culture Identification Culture separation and identification was performed in the food microbiology laboratory at Michigan State University. Reinforced clos- tridial agar (RCA) was used as a selective medium for isolation of L, bulgaricus, since it inhibits the growth of S. thermophilus. M17 broth 120 121 and agar media were used for isolation of S. thermophilus by suppressing the growth of L. bulgaricus. Morphological assessment was made for size, shape, arrangement and staining properties of cells. Pour plates and streak plates were made and incubated at 37°C for 24 h for S. thermophilus and 72 h for L. bulgaricis. To identify possible slime formation, dif- fering concentrations of sucrose in PCA were used. Various biochemical tests (Table 1) including litmus milk, salt tolerance, dye reduction (methylene blue) and acid production from various carbohydrate sources were used to aid in identification of yogurt bacteria. Sensory_Testing_ Initial sensory work defining preferred honey levels and processing parameters for later consumer evaluation were performed with the aid of eight trained dairy product judges. Statistical Methods A balanced incomplete block design (Type I where t = 6, k = 3, r = 10, b = 20) was used to evaluate the six (t) honey yogurt samples (Cochran and Cox, 1957, p. 472, Plan 11.5). Each panelist represented one incomplete block of three (k) samples. The basic plan was replicated five times giving 50 replications (5 x r) and 100 blocks (5 x b). The within-block sample order was balanced thus minimizing any positional effects. An analysis of variance for each attribute was carried out to deter- mine if any differences existed among the six samples. If a difference existed at the 95% level, the Duncan's Multiple Range test was employed to determine which samples were statistically different. 122 A multiple stepwise regression model was also computed to describe the relationship between overall acceptability and the four sensory attributes (color, sweetness, texture, and flavor). This analysis indi- cated which of the four attributes accounted for the most variability in the panelists' overall acceptability scores. Various statistical tables for this portion of the consumer evalua- tion are presented in Appendix I. The sensory score card is illustrated in Appendix II. Tasting and Scoring Each panelist tasted three of the six honey yogurt samples. The sam- ples were evaluated for color, sweetness, texture, flavor and overall acceptability. A semi-structured scale (Appendix II) was used to quantify the panel- ists' responses for each sensory criterion. Scoring was accomplished by placing a vertical bar for each sample on a ten centimeter line which had been anchored with extreme descriptive terms for each attribute. The lines were later measured to give the intensity of the panelists' responses. 123 A WHOLE * WATER /| HONEYj MILK . (when added) YOGURT MIX 1 .5%fat; 12. 6%MSNF , [IPASTEURIZE [88;°C 40min _ 70.3mm»2 first stage HOMOGIENIZE 35.2K9/CM2 second Stage 60l°C 4 . CULTURE [coouio 2H1_1_s% _ FILL YOGURT AND INCUBATE I 42¢1°c tlil pH 4.5 COLD STORAGE 5°C Figure l. Yogurt processing scheme. RESULTS AND DISCUSSION Various biochemical (Table 1) and microbiological tests were used to confirm that the bacteria taken from commercial yogurt were actual yogurt bacteria §, thermophilus, L, bulgaricus. Tests performed indicated that the bacteria used to manufacture buckwheat honey yogurt were S, 3335997 phjlu§_and L, bulgaricus. No slime formation was observed for either organism on sucrose plates. Yogurt produced contained no added stabili- zers or polysaccharide material which could participate in a stabilization role. The processing scheme for production of low-fat plain and low-fat buckwheat honey yogurt is depicted in Figure 1. Low-fat plain yogurt was used through this study as a control for both set time (time required to clot yogurt mix) and as a means to discern flavor defects related to cul- ture organisms or ingredient formulation. For all yogurts examined, pH decreased less than 0.1 pH after four days storage (5°C). For determination of desirable buckwheat honey levels 3.6 kg lots of plain (0%), 5%, 8%, 12% and 15% by weight buckwheat honey formulations were processed as previously described. Honey was added before pasteuri- zation. From Table 2 it is apparent that set time increased directly with concentration of honey. Buckwheat honey additions of 8% and above required 12 h or longer to set. Trained judges reported control yogurt (no honey addition) to have good typical yogurt flavor and good body and texture. All judged (8 exper- ienced individuals) reported 5% buckwheat honey yogurt to have good flavor and good body and texture. However, honey levels of 8% and above were found to be too sweet, as well as having poor body and texture. 124 125 Table 1. Biochemical Tests to Aid in Identification of Yogurt Culture Bacteria. Test S, thermophilus. L, bulgaricus Litmus Milk + + Salt tolerance 1% NaCl + 2% NaCl - Skim milk: With 0.01% MB Without MB + Acid production: Glucose Lactose Sucrose Maltose Mannitol Rhamnose lll+++ ll++ Growth at temperature of: 45°C + + 55°C 10°C Motility and nitrite production - 126 Table 2. Initial Evaluation of Buckwheat Honey Addition to Low-fat Plain Yogurt. Buckwheat Honey Addition (%) O 5 8 12 15 Yogurt mix pH 6.50 6.30 6.20 6.20 6.15 final pH 4.40 4.60 4.43 4.60 - set time 3.75h 3.75h 12 h a49d b- aGel formation observed at weekly intervals. bNo gel formation after 12 h incubation (43°C) and two months cold storage. 127 Because of time and energy requirements necessary for proper set of 8% honey yogurt as well as previously noted sensory panel criticisms lower levels of honey were then evaluated to define desirable levels of buckwheat honey. In another phase of the study lower levels of added honey (5%, 6%) were incorporated into the mix before pasteurization (BP) or after pas- teurization (AP) and then evaluated for flavor and body and texture by the experienced judges (Table 3). When honey was added before pasteur- ization set times were 3.5 h for 5% honey addition and 4.0 h for 6% honey addition, whereas when honey was added after pasteurization time required to set was doubled. Six of eight judges reported superior flavor for BP honey hogurt, while the other two judges felt that AP honey yogurt to be superior in flavor, while noting that yogurt processed this way had a coarse body and texture. Both honey levels were liked equally well by the trained panel of judges, Scale up processing operations (3.6 kg to 13.6 kg) were made to pro- vide for larger quantities that would be needed for consumer evaluation. Six 13.6 kg lots of yogurt (3 lots 5% honey, 3 lots 6% honey) were made and packaged in 4 oz (112 9) plastic containers with plastic lids stored for 2 d and then transported to Guelph, Ontario, Canada, for consumer evaluation. A balanced incomplete block design was used in the evaluation of six honey yogurt samples (three lots with 5% buckwheat honey and three lots with 6% buckwheat honey). Each sample was evaluated by 50 people (100 panelists tasted three out of the six samples) for color, sweetness, texture, flavor, and overall acceptability. The samples received similar 128 ratings for color and texture. The 6% honey yogurt was generally rated higher than the 5% honey yogurt for sweetness, flavor, and overall acceptability of the samples (Tables in Appendix I). 129 Table 3. Effect of Buckwheat Honey Added to Yogurt Mix Before (BP) and After Pasteurization (AP) as Related to Set Time. Honey Addition (%) O 5 6.5 Treatment none BP AP BP AP Initial pH 6.38 6.24 6.30 6.20 6.30 Final pH 4.25 4.32 4.42 4.38 4.60 Set time (h) 3.5 3.5 7.5 4.0 7.5 SUMMARY Lowfat honey yogurt is a nutritious and "natural" food that has lower caloric content than typical fruited yogurts. Six percent buck- wheat honey added to plain lowfat yogurt with no added stabilizers was preferred over the 5% addition. This product could be easily made in the home or on a commercial scale. Honey added before pasteurization was preferred by experienced dairy judges. When honey was added at culturing an inhibitory effect was noted and yogurt set time was doubled. Beck (J938) reported that bees may inject or spray a venom-like material that has an antifermentative and preserving effect in the honey comb. 130 LITERATURE CITED Beck, B.F. 1938. Honey and Health. Chapter 3. Robert McBride and CO. ’ NoYo Brown, D.G. and F.V. Kosikowski. 1970. How to make honey yogurt. Am. Dairy Rev. 32(4):60-62. Duthie, A.H., K.M. Nilson, H.V. Atherton and L.D. Garrett. 1977a. Proposed score card for yogurt. Cult. Dairy Prdts. J. 12(3):lO-12. Duthie, A.H., S. Wulff, K.M. Nilson, H.V. Atherton and L.D. Garrett. 1977b. Flavor panel selects all-natural maple-flavored yogurt. Cult. Dairy Prdts J. 12(4):8-lO. Kosikowski, F.V. 1977. Cheese and Fermented Milk Foods. Chapter 6. Yogurt. Edwards Bros. Inc., Ann Arbor, MI. 131 CHAPTER IV PHYSICAL DAMAGE OF YOGURT - THE ROLE OF SECONDARY PACKAGING ON STABILITY OF YOGURT] 1Presented at 1981 Institute of Food Technologists Annual Meeting. To be published in J. Food Science. 132 INTRODUCTION Commercial yogurt is packaged and distributed in a variety of ways (Table l) and many factors are involved in physical damage (phase separation and broken coagula) of yogurt, including agitation during transportation and handling (Rasic and Kurmann, 1978). Other factors include over acidification, low solids content, admixture of air, temperature fluctuations and various stabilizer additions. Neilson (1975) and Rasic and Kurmann (1978) reported that some stabilizers actually caused decreased acid production and increased phase separation. Good quality yogurt can be made without the use of commercial stabilizers, although the product is then more vulnerable to stress. Whey separation (syneresis) of yogurt is a common defect and should be controlled. Desirable firmness without syneresis is essential for a quality product (Kroger, 1976). This type of product damage can be caused by the commercial distribution environment. Vibratory motions are encountered by packaged products during shipping and distribution (Ostrem and Godshall, 1979). Vibration is common to all modes of transportation; and most products will be subjected to some vibration during shipment (MTS, 1976). There are no economically feasible means to completely eliminate the sources of vibratory motions during transportation. Therefore, it is necessary to design products and packages that will withstand vibration without a loss in product quality, while at the same time, minimizing packaging expense. 133 134 While actual evaluation of the product-package system throughout shipping is the most desirable means of testing packaged products, it is usually difficult or impossible to collect data in this manner. Therefore, laboratory test methods must be used to reduce overall evaluation time and expense. Both sour cream and yogurt in the retail shelf often show evidence of syneresis of whey, therefore, a series of trials was designed to observe the effects of vibration at the resonance frequency of yogurt products using different commercial distribution packages. Various shippers and overwrap systems were evaluated. Because little work has been performed assessing the role of 'packaging in influencing product damage in yogurt (Jones, 1980), this study was made to evaluate various commercial shippers of differing structural design on the quality of plain yogurt subjected to vibrations common in the shipping and distribution environment. Other considerations included in this research were impact shock testing and the effects of stabilizer addition, shipper perfonmance during incubation, cold storage and overwrapping. MATERIALS AND METHODS Yogurt Processing Lowfat plain yogurt mix was standardized to 1.5% fat, 12.6% solids not fat (SNF). The mix was pasteurized at 88°C for 40 min then cooled to 60°C and homogenized at 70.3 kg/cm2 first stage and 35.2 kg/cm2 second stage. The mix was cooled to 43°C and a mixed strain yogurt culture added (2% inoculation) to ripen the 135 product. Yogurt was packaged in 2279 waxed paper containers with plastic lids, incubated at this temperature to pH 4.5 and placed in a cold room (5°C) for 2 days. Resonance search and dwell testing was then performed on primary (plastic body and waxed paper) and secondary (shipper) containers. Experimental Using an MTS electrohydraulic vibration table a frequency search (3-40 Hz) was made for the following shipping containers (12 cups/shipper; stacked 10 high). I. Preformed molded pulp trays individually shrink wrapped with 1 mil polyethylene (PE); II. Wax coated paperboard trays with no film overwrap; III. Corrugated fiberboard sleeves individually shrink wrapped with 1 mil PE; IV. Corrugated fiberboard sleeves stretch wrapped over entire stack. After establishing reasonance, the stacks were vibrated at this frequency (constant input 0.59) for 15 min (dwell time). All stacks were then stored in the cold room at 5°C. After 8 h the yogurt was initially evaluated for product damage. All yogurts were evaluated on day 2, 5 and 10 of processing. Syneresis or whey-off was indicated qualitatively by: - no whey-off; :_very slight; + slight; ++ definite. At the termination of storage the extent of syneresis was quantitated by collecting and weighing the free surface whey using a Mettler P1210 balance. Filled yogurt containers (waxed paper containers with plastic snap on lids) were evaluated for impact damage using an MTS model 846-240 impact shock machine. Samples were tested in duplicate. 136 02000.0 020 .02220.0 0%”..mm 20.0000-0. 22.220 0..00.. 00.0 22 .000 0..00.. 2..2 0.00.0 ..02 0.02.0 0.0..-0 02000.0 0..00.. N0 - .0.20.0 00 0002200 .000 20.0. 0.202 x... 0 N. ....0. .0... 0.0 ..0% ..02.0 0 . wm0m00w.m«mm 20....0-0. .02. 0.00.0 02mwmwwmxm 20.2 0 0000 02000.0 020 .2002. 20.0.00-0 2..z 2. 00200. .000 0..00.. .02. .00 .00 0.02.20. 00202 20.2 0 0000 02000.0 020 0200020... 20....0-.. 2..2 2. 00200. .000 20.0. 00202 .02. .00 .00 0020220 02000.0 ..0. 0.0..-. 00 - 200 0.0.200. 00.0002200 .000 0..00.. 20.. 0 0. ..02 0.02.0 m2:mo.o 0:0 .oom 00 n , :o no a x¢(3-¢A WN-fl 80¢.- ZG-flmfl Swat-39 J<=umh¢a CZ—Q‘XO“ .352; 3.2.8800 0.3.3:“. .3 2053.30 9.39... .0 3333.09.20 I p 03d... 137 r M TOP VIEW FRONT VIEW MTS VIBRATION TABLE Figure 1. MTS e1ectrohydrau1ic vibration table. 138 Vibration Table The electrohydraulic system consists of a control console and a vibration table (Figure 1).. The vibrator maintains a constant input across a broad range of frequencies and the test specimen (yogurt stack) amplifies the input at critical frequencies (Moore, l976). Such equipment enables one to ascertain resonance, which is the frequency where the acceleration or amplitude is maximal. At this point, any adjustment of frequency will reduce the amplitude or strength of vibration. Gordon and Bains (l979) concluded that most unit loads have major resonance frequencies between 7 and 30 Hz. This type of testing procedure employs a sine sweep and dwell over a predetermined range.(3-40 Hz) to determine resonance points. The frequencies of concern are then held (dwell) for a period of time to determine the likelihood of damage. For evaluation of effects under practical commercial shipping conditions at least one stacked column of containers is tested (MTS, l976). In a recently published search of yogurt storage and packaging covering l972-79, no work on vibration or shock testing was reported (Jones, 1980). Vibration during shipment of other food products has been related to product damage. RESULTS AND DISCUSSION Resonance vibration for primary containers, waxed paper and plastic, occurred at 22 and 24 H2, respectively. Resonance for shippers I-IV occurred at approximately ll Hz. After vibration, l9, l6, 38 and 16 percent of the primary containers in shippers I-IV, respectively, showed slight or definite whey-off. Ten days after processing 56, 39, 30 and 13 percent of the primary containers 139 exhibited whey-off. The bar graph in figure 2 illustrates the comparison between shipper types and phase separation on day two and day ten of processing. At the end of the storage period the extent of syneresis was quantitated. Slight whey-off corresponded to 0.2% - 0.6% (w/w) whey and definite whey-off from 0.6 - l.8%. Control samples which were not vibrated exhibited minimal or no whey-off during storage, and pH values decreased 0.1 unit during this period. At the conclusion of the storage study, definite whey-off was visible in l0-20 percent of the samples in shippers I-III. Less than l percent of the samples in shipper IV (stretch wrapped) showed definite whey-off. This shipper also showed least overall damage. Twenty five and l4 percent of the samples in shippers II and III appeared to have broken (cracked) coagula, whereas in shippers I and IV this type of damage was minimal ( six). Most damage, for all shippers, occurred in the top layers of the stacks. Stretch wrapping considerably minimized this type of damage. Statistical analysis (Gill, 1978) revealed that there was a significant difference between shipper types (I-IV) and resulting product damage (Table 2). Individual comparisons using Bonforroni Chi-square statistics showed shipper IV (stretch wrapped corrugated fiberboard sleeves) to have less damage (p $0.0l) than the other three shippers tested (1, II, III). The most dramatic difference was between shipper IV and II. No significant differences (P <0.05) between other shipper comparisons was noted (II vs I, II vs III, 111 vs 1). Using this type of testing, various materials and processing parameters could be evaluated for their effectiveness in minimizing whey-off or product damage during distribution. 140 TABLE 2. Statistical analysis of physical damage1 occuring in yogurt vibrated in selected shippers. SHIPPER COMPARISON I - IV IV vs I IV vs II IV vs III III vs I II vs III II vs I VII - VIII TEST STATISTIC 45.72 23.29 47.44 29.55 5.534 3.278 0.575 l.690 SIGNIFICANCE Pvvmchx H 00a.m 0. 0030.0 3mm=0 0o1 man: >0010a000. 000000000 2. 00 00 0. 00 00 0.0.. Ama :ozmkv Amx zozmkv 00.00 0.0. 0.00 0.00 0.0. 0.0. 0.0. 0..0 020000000 0... 0.0. 0...0 0.00 0.00 0.000 0.00 00.000. 0.0. 0.0. 0... 0.0. 0.0. 0... 0.00 0.0000 ..0. ..000 0.000 0.00. 0.00 0..00 0.00 o