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LIBRA Fir * Michigan Stan: .. .* ‘Unimt‘y This is to certify that the thesis entitled STABILITY AND SENSORY PROPERTIES OF YOGURT DRINK presented by Seung Tak, Hong has been accepted towards fulfillment of the requirements for M.S. degreein Food Science W Major professor Datemmo 0-7639 OVERDUE FINES: ...._______.__ . 25¢ per day per item RETURNING LIBRARY MATERIALS: P lace in book return to arge from circulation records ‘14,.“ ”adv!“ AUG 2 6 2005 STABILITY AND SENSORY PROPERTIES OF YOGURT DRINK BY Seung Tak Hong A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1980 ABSTRACT STABILITY AND SENSORY PROPERTIES OF YOGURT DRINK BY Seung Tak Hong The effect of variations in processing parameters and in the concentration of stabilizers on the stability and sensory properties of yogurt drink was investigated. Yogurt drink was composed of 25% plain low fat yogurt, 10% frozen concentrated orange juice, 9% sugar and 56% water. Stability studies were carried out by observing sediment or syneresis volume of the samples during storage at 5°C and by determining protein content on the supernatant fluid of the samples after storage. Subjective sensory analysis was conducted using a nine- point hedonic scaling method or ranking method. Of four pH values (3.2, 3.6, 4.0 and 4.2), pH 4.0 was the best for product stability and flavor. Heat treat- ment variation (65°C/30 min, 75°C/10 min, 88°C/2 min and 115°C/4 sec) and homogenization pressure variation (70 kg/cmz, 105 kg/cmz, 14o kg/cm2 and 175 kg/cmz) had little effect on the sensory properties of the product. High temperature pasteurization and low pressure homogenization favored stabilization of the product. Seung Tak Hong Among the stabilizers studied, carrageenan, propylene glycol alginate and pectin were not effective in stabilizing the product. Guar gum and a commercial preprietary stabilizer were very effective, stabilizing the product at the concentra- tions of 0.20% and 0.30% respectively. The use of guar gum in combination with alginate, pectin or carboxymethyl cellulose did not show the synergistic effects of the com- bined stabilizers. The stabilized samples showed excellent acceptability in taste panel evaluation. ACKNOWLEDGEMENTS The author expresses his sincere gratitude to Dr. C. M. Stine for his help and counsel during the course of this research and preparation of the manu- script. Grateful acknowledgement is due members of the guidance committee: Dr. R. C. Chandan and Dr. P. Markakis, Department of Food Science and Human Nutrition, Dr. H. A. Lillevik, Department of Biochemistry for their advice and effort in reading this manuscript. The author is also indebted to his friends and colleagues for their invaluable help and advice. It is with the utmost gratitude that this thesis .is dedicated to the author's parents for their encour- agement and moral support throughout the entire study. The author is also most grateful to his wife, Chang, for her devotion, patience and understanding. ' ii TABLE OF CONTENTS ACKNOWLEDGEMENTS . . . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . Historical Aspects of Yogurt Products Yogurt Technology - Plain Yogurt . . Standardization and Treatment of Yogurt Mix . . . . . . . . . . . . . . . Standardization . . . . . . . . . Pasteurization . . . . . . . . . Homogenization . . . . . . . . . Microbiological Aspects of Yogurt . Propagation of Cultures . . . . . Inoculation and Incubation . . . Flavor Development in Yogurt . . Cooling and Storage . . . . . . . . Yogurt Drink . . . . . . . . . . . . Processing . . . . . . . . . . . . Future of Yogurt Drink . . . . . . MATERIALS AND METHODS . . . . . . . . . . Preparation of Samples . . . . . . . Preparation of Mother Culture . . . Culture . . . . . . . . . . . . . Culture Medium . . . . . . . . . Devices for Transfers . . . . . . Culture Room . . . . . . . . . . iii Page ii vi ix \DxIU'IU'I 10 14 16 19 22 23 24 27 29 29 29 29 29 29 29 Incubation . . . . . . . . . . Preparation of Yogurt . . . . . Treatment of Milk . . . . . . Incubation . . . . . . . . . . Cooling and Cold Storage . . . Preparation of Yogurt Drink . . Composition . . . . . . . . . Processing . . . . . . . . . . Cooling and Cold Storage . . . Determination of Fat and Total $0 Yogurt and Yogurt Drink . . . Experimental Variables in the Prepa Yogurt Drink Samples . . . . . . Processing Parameters . . . . . pH.............. Pasteurization Temperature/Time Homogenization Pressure . . . Stabilizers . . . . . . . . . . Stability Studies . . . . . . . . Sediment . . . . . . . . . . . . Syneresis . . . . . . . . . . . Protein Determination . . . . . Sensory Evaluation . . . . . . . . Ranking Method . . . . . . . . . Hedonic Scaling Method . . . . . Determination of Optimum Processing Parameters O O O O O O O I I I I pH 0 O C O O O O I C O O O O O O Pasteurization Temperature/Time Homogenization Pressure . . . . Determination of the Most Suitable lids in ration of Stabilizers and their Concentrations in Yogurt Drink . . RESULTS AND DISCUSSION . . . . . . . . Composition of Yogurt and Yogurt Dr Chemical and Physical Properties of Drink O O O O O O O O O O O I 0 iv ink . . . . Yogurt Page 29 30 30 30 30 30 30 32 32 32 34 34 34 34 34 34 35 35 36 36 37 37 38 39 39 39 39 43 44 44 47 Page Effect of Processing Parameters on the Sta- bility and Sensory Properties of Yogurt Drink. . 50 pH 0 O C O O O O O I O O O O O O O I O O O O O I 50 Pasteurization Temperature/Time . . . . . . . . 57 Homogenization Pressure . . . . . . . . . . . . 61 Effect of Stabilizers on the Stability and Sensory Properties of Yogurt Drink . . . . . . . 67 SWRY AND CONCLUSION O I O O O O O O O O O O O O O O 8 3 APPENDIX Statistical Chart 0 O O O O O O O O O O O O O 86 LIST OF REFERENCES 0 O O O O O O O O O O O O O C O O C 90 Table l. 10. LIST OF TABLES Suggested standards for the chemical composition of yogurt in terms of milk fat and SNF . . . . . . . . . . . . . . Effect of temperature on homogenization of yogurt mix . . . . . . . . . . . . . Some methods currently employed for the inoculation and incubation of yogurt cultures 0 O O O O O O O O O O C O O O Treatments of yogurt drink to determine optimum pH on the stability and sensory properties of the product . . . . . . . Treatments of yogurt drink to determine optimum pasteurization temperature/time on the stability and sensory properties of the product . . . . . . . . . . . . Treatments of yogurt drink to determine optimum homogenization pressure on the stability and sensory properties of of the product . . . . . . . . . . . . Chemical composition of yogurt and yogurt drink . . . . . . . . . . . . . Ranked scores of yogurt drink at the sugar concentrations of 7%, 8%, 9% and 10% O O O O O O I O O O O O O O O O O 0 Scores of the samples used to study the effects of pH on the sensory properties of yogurt drink . . . . . . . . . . . . Analysis of variance from the data of Table 9 . . . . . . . . . . . . . . . . vi Page 11 17 40 41 42 45 46 52 53 Table 11. 12. 13. 14. 15. l6. 17. 18. 19. 20. 21. Page Difference of sample means from the data Of Table 9 O O O O O O O O O O O O O O O O 53 Sediment volume of the samples used to study the effect of pH on the stability of yogurt drink . . . . . . . . . . . . . 55 Protein content of supernatant fluid of the samples used to study the effect of pH on the stability of yogurt drink . . . . . . 56 Scores of the samples used to study the effects of pasteurization temperature/time on the sensory properties of yogurt drink. 58 Analysis of variance from the data of Table 14 O O C O O O O O O I O O O O O O O 59 Ranked scores of the samples used to study the effects of homogenization pressure on the sensory properties of yogurt drink . . . . . . . . . ... . . . . 62 Sediment volume of the samples used to study the effect of homogenization pressure on the stability of yogurt drink . . . . . 64 Protein content of supernatant fluid of the samples used to study the effect of homo- genization pressure on the stabiity of yogurt drink . . . . . . . . . . . . . . . 65 Syneresis volume (ml) of yogurt drink samples after storage as a function of stabilizers at different concentrations. pH; 4.0, heat treatment; 88°C/2 min, homogenization pressure; 175 kg/cmz. . . . 69 Syneresis volume (ml) of yogurt drink samples after storage as a function of stabilizers at different co centrations. pH; 4.0, heat treatment; 88 C/g min, homo- genization pressure; 175 kg/cm . . . . . 70 Syneresis volume (ml) of yogurt drink samples after storage as a function of stabilizer blends at different concentra- tions. ph; 4.0, heat treatment; 88°C/2 min, homogenization pressure; 175 kg/cm . 78 vii Table 22. 23. 24. Page Scores of the two samples finally stabilized by the use of stabilizers at the designated processing parameters . 81 Rank totals required for significance at the 5 percent level . . . . . . . . . . . 86 Variance ratio - 5 percent points for distribution of F . . . . . . . . . . . . 88 viii Figure 1. 2. LIST OF FIGURES Page Flow diagram of yogurt manufacture . . . 31 Flow diagram of yogurt drink manufac- ture O O O O O O O O O O O O O O O O O O 33 Syneresis volume of yogurt drink sam- ples after storage as a function of different concentrations of carrageenan. pH; 4.0, heat treatment; 88°C/ 2 min, homogenization pressure; 175 kg/cm . . 71 Syneresis volume of yogurt drink samples after storage as a function of different concentrations of gugr gum. pH; 4.0, heat treatment; 88 C/2 min, homogenization pressure; 175 kg/cm2 . . Syneresis volume of yogurt drink sam- ples after storage as a function of different concentrations of commercial proprietary blended stabilizer. pH; 4.0, heat treatment; 88 9/2 min, homogenization pressure; 175 kg/cm . . . . . . . . . . Syneresis volume of yogurt drink sam- ples after storage as a function of different concentrations of propylene glycol alginate. pH; 4.0, heat treat- ment; 88°C/2 min, hemogenization pressure; 175 kg/cm . . . . . . . . . . Syneresis volume of yogurt drink samples after storage as a function of different concentrations of pectin. pH; 4.0, heat treatment; 88°C/2 mi , homogenization pressure; 175 kg/cm . . ix 72 73 74 75 INTRODUCTION The tremendous increase in yogurt consumption in the United States and all over the world has led to the development of other yogurt based dairy products as an opportunity for product expansion. Yogurt drink has been introduced as a product with a refreshing taste of liquid drink combined with the natural ingredients of yogurt. Yogurt drink is somewhat unstable because of its high solids content (usually higher than 15.0%) in liquid medium and the physical instability of the milk proteins at low pH. Therefore, the problem is to pro- duce a finished product that is stable and has the necessary sensory characteristics to make it acceptable to the consumer. A few general trade journal articles have described yogurt drink and suggested composition and manufacturing procedures. However, little research appears to be available on the effects of processing parameters and the functions of stabilizers on the stability and sen- sory properties of the product. In the present in- vestigation, a step by step approach to the stabilization of the product is employed using four variations in pH, heat treatment and homogenization pressure respectively. Six stabilizers are investigated individually and in combinations at various concentrations. Subjective 1 sensory analysis is conducted in each step prior to the stability studies which include sediment or syneresis observation and protein determination on the super- natant fluid of the samples. The present study was undertaken to establish op- timum processing parameters and the concentration of suitable stabilizer(s) on the stability and sensory properties of yogurt drink. This approach to the problem offers the possibility of developing information applicable to the control of processing parameters and to the use of stabilizers to maximize the stability in yogurt based drinks. LITERATURE REVIEW Historical Aspects of Yogurt Products Yogurt is a cultured milk product that is made by in— oculating warm pasteurized and/or homogenized milk with a combined culture of S. thermophilus and L. bulgaricus (In- ternational Dairy Federation, 1969). The word yogurt (also spelled yoghurt, yourt or yoghourt) is of Turkish origin. The yogurt or yogurt-like products dates back to Biblical days--or to when man first began drinking the milk of cows, goats, sheep and camels, although there is no precise record when they were first made. In the Bible, it is recorded that Abraham served sour and sweet milk along with a calf while entertaining three angels (Genesis 18:8). It is believed that every civiliza- tion on earth has known and consumed fermented milk of one type or another. Many of these foods can still be found throughout the world, such as buttermilk in Europe, acido- philus milk in the Soviet Union, yogurt in Turkey, dahi in India, leben in Egypt, yiaourti in Greece, cieddu in Italy, kefir in Balkan countries, and kumiss in Mongolia (Kosikow- ski, 1977; Rasic and Kurmann, 1978). Originally yogurt was made from the milk of sheep, goats, and cows. The propagation was carried out by using a small quantity of the previously fermented milk to seed the next container of milk. Modern interest in yogurt owes 4 much to the work of Metchnikoff, a Russian born bacteriolo- gist who worked at the Pasteur Institute in Paris (Tramer, 1975; Davis, 1974; Rasic and Kurmann, 1978). The charac- teristic organism of the traditional soured milk of the Balkans was described and named as Lactobacillus bulgaricus. The good health and longevity of the Balkan peasants were attributed to the effect of this organism in the intestinal tract through repressing the undersirable putrefactive bacteria. Although this theory exaggerated the value of yogurt, it significantly influenced the spread of the pro- duct to many countries of EurOpe and promoted extensive studies by subsequent workers. Since World War II, an increased knowledge of the science of microbiology, better understanding of the fac- tors affecting the sensory properties of yogurt and the application of modern equipment have resulted in the rapid advancement of yogurt technology. The introduction of fruit and the application of a wide range of flavors pro- moted further growth in the consumption of yogurt products. The United States has commercially produceélyogurt only since 1940. The first fruit flavored yogurt was in- troduced in the United States in the early 1960's and con- sumption has increased at a steady pace since then. According to the annual report of the Milk Industry Foun- dation (1979), yogurtconsumption has increased from 0.11 lbs per capita in 1955 to 2.68 lbs per capita in 1978, an increase of 2,500%. Yogurt Technology - Plain Yogurt Yogurt is similar to other cultured dairy products in that high quality ingredients and strict attention to sanitary procedures are required for satisfactory manu- facture of an acceptable product. There are certain basic principles to be followed regardless of the type of yogurt made. Standardization of milk, pasteurization temperature and time, homogenization, time and temperature of incubation and handling of the product after manufacture are important steps in the process. Standardization and Treatment of Yogurt Mix Standardization - There are no concrete regulatory standards of composition as to fat and SNF contents of yo- gurt (Duitschaever et al., 1972; Kroger and Weaver, 1973; Robinson and Tamime, 1976; Richmond et al., 1979), as shown in Table 1. It has been common practice for the dairies to experiment with fat and solids not fat (SNF) content of yogurt by the application of available knowledge and expertise, and to modify its composition as desired. Stan- dardization, not only insures consistency of the end product but also enables control of the flavor and aroma, viscos- ity, stability and nutritive value of the finished product. Kroger (1973, 1976) and Davis (1974) stated that milk fat in yogurt mix definitely contributes to the smoothness of mouthfeel and richness of the product if the mix is Table 1. Suggested standards for the chemical composition of yogurt in terms of milk fat and SNF. Country Types of yogurt based of origin on % fat % SNF Normal Medium Low Denmark 3.8 1.8-1.5 0.3 ----- France 3.0 ----- 1.0 ——--« Netherlands 3.2 ----- 0.3 ----- United Kingdom 3.5 2.0-1.0 0.3 8.5 United States 3.8-3.0 2.8-2.0 1.0—0.5 8.25—8.3 FAQ/WHO 3.0 3.0-0.5 0.5 8.2 Source: Robinson and Tamime, 1976. homogenized. Nielsen (1975) stressed the importance of fat in relation to both body and texture of yogurt. It was indicated that even 1.0-2.0% fat in yogurt has a bene- ficial influence on body and texture of the product. Rasic and Kurmann (1978) observed that skimmilk yogurt is more acid, less mild and less aromatic than yogurt which con- tains milk fat. It was further stated that the fat con- tent of 1.5% can counteract the above defects. It is usual in practice to increase SNF content in milk for yogurt by concentration of the milk or by addition of nonfat dry milk (NFDM). Powell (1970) and Davis (1971) considered that NFDM added at the rate of 1 to 2% gives optimum solids content for yogurt mix, yielding afirmer gel and no syneresis in the finished product, and contri- buting to greater nutritional value. According to Cottenie (1978), greater improvements in quality can be obtained if the increase in solid concentration is brought about by partial concentration under vacuum, than if NFDM is added. A change in the ca1cium-phosphorus-caseinate com- plex induced by the removal of water is beneficial for building up an optimum micelle structure of proteins which reduces and eliminates the tendency for syneresis. Excessive addition of SNF gives rise to defects such as saltiness, powder flavor and coarseness in the end product. Pasteurization - Heat treatment of yogurt mix is carried out with the purpose to kill pathogenic organisms which may be present, to produce an improved medium for the growth of lactic acid bacteria, and to improve consistency and firmness of the product (Powell, 1970; Sellars, 1973; Nielsen, 1974, 1975; Davis, 1975; Cottenie, 1978; Rasic and Kurmann, 1978). The improved medium for the growth of the lactic acid bacteria is attributed to a reduction in bacteriostatic substances and the formation of substance which act as stimuli to the growth of the culture. Davis (1975) sug- gested that heating lowers the oxygen tension and makes a more favorable medium for the growth of L. bulgaricus. Heat treatment may also liberate amino acids from the milk proteins which may stimulate the growth of L. bulgaricus. This organism in turn elaborates amino acids which stimu- late growth of S. thermgphilus (Pette and Lolkema, 1950 a and b; Nielsen, 1975). Nielsen (1975) suggested that the improved consistency and firmness of yogurt made from heated milk is associated with the denaturation of whey proteins, the change of caseinate structure and the association of these two phe- nomena which increase the water-binding capacity of the system. Cottenie (1978) reported that accumulative heat treatment of yogurt mix at 190°F for 2 min followed by an additional 192°F for 3 min results in good consistency and firmness of yogurt. Galesloot and Hassing (1968) recommended heating of yogurt mix to 185°F for 30 min for firm consistency. Nielsen (1974) suggested that the best texture can be obtained with a heat treatment of 195°F for 5-30 min. Rasic and Kurmann (1978) explained that the differences in the data reported by many investigators may be due to the probable effects of other factors on the hydrOphilic properties of porteins in addition to heat treatment of yogurt mix, e.g. the total solids content and particularly the protein content of milk, the solid content adjustment of milk, the properties of yogurt cultures, homogenization, etc. Niel- sen (1975) claimed that a yogurt mix with high SNF con- tent should be heated at 195°F for not less than 10 min and not more than 50 min. Generally, yogurt mix is pasteurized at temperatures of 190°F, holding times being between 5 and 45 min. (Humphreys and Plunkett, 1969). Homogenization - Pette and Lolkema (1951 b) maintained that homogenization is essential for the production of firm curd yogurt. Cottenie (1978) noted that homogenization, if carried out efficiently, is quite necessary because it prevents fat separation, increases the firmness of the gel by changing the physical properties of fat globules and casein particles, improves the taste through the formation of a soft, finely homogeneous curd and shortens the curdling time. Pette and Lolkema (1951 b) recommended that pasteuri- zation be carried out before homogenization to prevent 10 lipolysis caused by lipolytic enzymes. However, Rasic and Kurmann (1978) stated that this procedure has a limitation because of the risk of recontamination. Rasic and Kurmann (1978) recommended high pressure homogenization, claiming that high pressure prevents rising of fat by breaking up the fat globules to sufficient small size and increases viscosity of yogurt by denaturing more'serum proteins. On the other hand, Humphreys and Plunkett (1969) maintained that homogenization at a low pressure significantly in- creases firmness and viscosity and reduces the rate of whey separation on storage. These investigators, quoting from personal communications from Barker (1965), also mentioned that good quality yogurt can be produced without homogeniza- tion. Stocklin (1969) reported that homogenization at 110°F at a pressure of 1400 or 1500 psi produced a good yogurt. Rasic and Kurmann (1978) presented the effect of tem- perature on homogenization of yogurt mix as shown in Table 2. The temperatures shown differ depending on the purpose of the homogenization. Microbiological Aspects of Yogurt There is considerable confusion in terminology in the literature as to the microorganisms that are used for the manufacture of yogurt. However, most workers agree that yogurt is basically cultured milk fermented by L. bulgaricus growing in association with S. thermophilus (Pette and Lolkema, 1950, 1951; Stocklin, 1969; Humphreys 11 Table 2. Effect of temperature on homogenization of yogurt mix. Temperature Effect on milk constituents 100°F Minimum temperature that can be used. 122-1400F Optimum condition. 140-1580F Damaging the ability for fat rising. Above 158°F Serum protein begins to be denatured. Above 212°F Caseins begins to coagulate when temperature combined with a sufficient pressure. Source: Rasic and Kurmann, 1978. an l9 19 cu; re; t9] cia opt dif 685 m gro 12 and Plunkett, 1969; Powell, 1970; Angevine, 1972; Tramer, 1973; Sellars, 1973; Robinson and Tamime, 1975; Kosikowski, 1977; Cottenie, 1978; Lundstedt, 1978; Sharpe, 1979). Yogurt cultures are best obtained from established culture manufacturers, from research institutes or other reputable sources and should be renewed at frequent in- tervals. Handling of yogurt cultures requires skill and appre- ciation of several facts, namely, that they have different optimal growth temperatures, that their rate of growth is different and that in spite of all these variations it is essential to maintain a balance of the two organisms. It is well established that L. bulgaricus and S. thermophilus stimulate each other during their associative growth. Pette and Lolkema (1950 a and b) proved the stimulation of S. thermophilus by L. bulgaricus. The study suggested that L. bulgaricus liberates in milk certain amino acids which stimulate S. thermophilus. In their futher investi- gations, it was shown that valine was the most important amino acid among those which stimulate the growth of S; thermophilus. Bautista et al. (1966) confirmed the stimulatory effect, but found that the active factors were glycine and his- tidine. 13 Galesloot et a1. (1968) showed that S. thermophilus also stimulates the growth of LL‘hnlgazigg§,by producing a factor that is equal to or can be replaced by formic acid and this stimulation can only be demonstrated in moderately heated milks. It was further pointed out that Pette and Lolkema (1950 a and b) and Bautista et a1. (1966) had failed to observe this stimulation by S. thermophilus because they had used steamed milk or sterilized milk which contains formic acid as a consequence of intensive heat treatment. Tramer (1973) showed that a mixed yogurt culture of both L. bulgaricus and S. thermophilus gave more acid de- velopment in milk heated to the usual time/temperature combination of 203°F for 30 min than in milk heated severely (autoclaved). Besides the symbiotic action, Pette and Lolkema (1950 a) demonstrated an inhibitory action of L. bulgaricus upon S. thermophilgio caused by the lactic acid produced. S; thermophilus, which are stimulated in the first stage by L. bulgaricus, predominate when the acidity of the culture is still low, while L. bulgaricus increase in number mainly in the later stage of the fermentation process, because they are more acid tolerant. According to Kroger (1973), Tramer (1973), Sellars (1973) and Davis (1975), S. thermophilus grew best at pH ’6.5, with growth stOpping at pH 5.2-4.4; L. bulgaricus grew best at pH 5.5, with growth stopping at pH 3.5-3.8. 14 Temperature of 110-1150F are ideal for optimal growth of L. bulgaricus, while S. thermophilus grow better at 90- 100°F (Angevine, 1972; Sellars, 1973; Davis, 1975). Pette and Lolkema (1951 a) found that during incubation the prOportion of both kinds of bacteria is influenced by the incubation time, the acidity of the culture and by the incubation temperature, as the optimum growth temperature is somewhat lower for S. thermophilus than for L, bulgari- cus. The concentration of S. thermophilus cells can be increased by lowering the incubation temperature, lowering the amount of inoculum or by incubating to a lower acidity. The converse of these conditions favors the increase of L. bulgaricus. Most investigators (Pette and Lolkema, 1951 a: Stocklin, 1969; Powell, 1970; Davis et al., 1971, 1974; Kroger, 1973; Sellars, 1973; Nielsen, 1975; Kosikowski, 1977) agree that the optimum ratio of S. thermophilus and L. bulgaricus at the end of incubation should be about 1:1. Prgpagation of cultures - The medium used for propa- gation of cultures should be clean and fresh, of low bac- terial content, and free from inhibitory substances. Spray-dried NFDM reconstituted to 10-11% solids has been claimed to be an excellent medium for the propagation of cultures (Davis, 1956; Sellars, 1973). Sellars (1973), Cottenie (1978), Rasic and Kurmann (1978) suggested that the reconstituted medium should be 15 heat treated in an autoclave for 15 min at 15 lbs steam pressure, or in a water bath at 190-2200F for 1 hr or in flowing steam for 1 hr since these heat treatments improve the medium as substrate for the bacteria by destruction of the initial organisms, destruction or inactivation of vegetative forms of spores, denaturation of whey proteins and by expelling dissolved oxygen. The exact temperature of incubation depends on the particular culture employed and balance of organism de- sired. The incubation time depends on the amount of in- oculum used and on culture activity. According to Sellars (1973), most cultures when in- oculated into the medium at the 1% concentration and incubated at 104OF cause coagulation in 6-8 hr. Lund- stedt (1969) and Angevine (1972) reported that lower incubation temperature of 86-900F results in better growth balance of L. bulgaricus and S. thermophilus. Cottenie (1978) advocated 110-113OF as an optimum temper- ature for cultures. Ashton (1963) advocated the use of separate culturing, claiming that this allows for a greater degree of flexibility and control. If the cul- tures are propagated spearately, the L. bulgaricus is usually inoculated at llOOF, while 8. thermophilus is inoculated at 100°F (Stocklin, 1969). To maintain optimum activity most cultures should be cooled to 40-450F when they have developed a pH of 4.0-4.5. 16 Inoculation and Incubation - The pasteurized and homogenized yogurt mix is cooled to the inoculation temper- ature and then inoculated with thoroughly mixed culture. Many investigators have reported and suggested dif- ferent amount of inoculum, temperature/time of incuba- tion and pH at the end of incubation. This reflects the differences in type and activity of the culture, compo- sition of yogurt mix, condition of the culture at the time of incubation, rate of cooling and the acidity of yogurt desired. The data in Table 3 indicate that the amount of culture used ranges from 1 to 5%, incubation temperature from 104 to 1130F, incubation time from 2 to 6 hr and final pH from 4.1 to 4.4. Ashton (1963) advocated incubation of S. thermophi- lus at 98-1000F to a low acidity, and of L. bulgaricus at 108-1100F to higher acidities and for longer times. At inoculation, the culture conditions can be varied de- pending on the relative acidity of the two cultures. It was claimed that the separate culture method is the more flexible and easier to control and tends to enable better control of variable physical characteristics which are less controllable by the mixed culture method. According to Lundstedt (1969, 1973) the best incuba- tion time-temperature combination for obtaining top quality product in terms of body, texture, and flavor is 86°F, for 17 Table 3. Some methods currently employed for the in- oculation and incubation of yogurt cultures. Amount . Origin of Temperature Time giggit Culture y Stocklin 1% 110°F 3-3 1/2 hr pH 4.3 (1969) Powell 2-5% 108 - 113°F 2-6 hr pH 4.1 (1970) Sellars 2% 104 - 113°F Depends on (1973) desired fi- nal acidity . o " Dav1s 1-2% 110 F (1973) Kroger 2% 110°F 3-3 1/2 hr pH 4.2 (1973) Kosikowski 2-5% 113°F 3-6 hr pH 4.4 (1977) Cottenie 2.2% 11o°F 4 hr pH 4.4 (1978) 18 14 to 16 hr. The advantages of the low temperature, long set method were pointed out; a) the proper ratio of bac- teria is obtained and as a result the symbiotic metabolic byproducts are balanced to give the ideal acid-acetaldehyde concentration, b) the body and texture are good and there is less tendency to wheying-off, c) cooling to prevent too much acid development can be achieved faster and it re- quires less energy input, and d) packaging could be scheduled more conveniently. Obert (1978) reported that prolonged incubation for 10—16 hr at 90-950F is the most effective and cheapest for improving yogurt consistency. However, Kosikowski (1977) pointed out that this low temp- erature, long set method requires markedly increased net time of processing and more critical control of temperature is necessary since this temperature range is near the point at which the culture organisms do not thrive. After inoculation, the symbiotic growth of two micro- organisms starts. 3. thermophilus grows rapidly until a pH of about 5.5 is reached. Then, the growth of L. bul- garicus is progressively favored. If incubation is not halted at between pH 4.0 to 4.4, the acidity would go well below pH 4.0 since L. bulgaricus is capable of growing at this low pH (Kroger, 1976). Normally 20 to 30% of the lactose is fermented, although greater fermentation has been reported (Rasic and Kurmann, 1978). Coagulation commences at pH of about 5.3, depending on the milk and 19 the protein content (Davis, 1956). At pH 4.6-4.7 pre- cipitation is essentially completed, with the caseins existing in a salt-free state (Humphreys and Plunkett, 1969; Rasic and Kurmann, 1978). Acid development can be monitored by titration or measurement of pH. Flavor Develgpment in Yogurt - Development of yogurt flavor goes hand in hand with acid production through a symbiotic bacterial relationship. The characteristic flavor of yogurt is due to lactic acid, which is odorless, and to trace amounts of acetaldehyde, diacetyl and acetic acid. The original milk components and their concentrations also play a role, especially the fat and SNF (Kroger, 1973; Sandine et al., 1974; Kosikowski, 1977). According to Pette and Lolkema (1950 c) the main flavor components of yogurt are lactic acid and an aroma substance produced by L. bulgaricus. The aroma substance from the cultures was distilled and acetaldehyde was found in the distillate. Greater amount of acetaldehyde was observed when the acidity was high. Keenan and Bills (1968) also emphasized the importance of this compound in yogurt flavor. They suggested that high concentration of acetaldehyde is necessary to produce a desirable flavor in yogurt. Bottazzi and Vescovo (1969) correlated acetaldehyde content with yogurt flavor. A good flavored yogurt 20 resulted from L. bulgaricus strains producing greater than 8.0 ppm acetaldehyde and weak flavor resulted when less than 4.0 ppm of acetaldehyde was produced. These workers also suggested that acetone may be involved in typical yogurt flavor. A ratio of 2.8:1 (acetaldehydezacetone) produced a strong yogurt flavor. Hamdan, et a1. (1971) have ascertained levels of 23-26 ppm acetaldehyde in natural yogurt, while 15-20 ppm was indicated as a normal range by Bottazzi and Vescovo (1969). Kroger (1976) also stressed the importance of acetaldehyde for good yogurt flavor, indicating that acetaldehyde comprises about 90% (50 ppm) of the carbonyl compounds present in yogurt. Rasic and Kurmann (1978) considered that the optimum flavor of yogurt is obtained by an acetaldehyde content ranging between 23 and 41 ppm. Bottazzi and Dellaglio (1967) considered that acetal- dehyde and diacetyl are important flavor components of dairy products. The production of these two compounds by S. thermophilus and other lactic streptococci was inves— tigated. It was shown that S. thermophilus formed more acetaldehyde and diacetyl than other homofermentive lactic streptococci studied. Lindsay and Day (1965) found that the concentration of acetaldehyde and diacetyl in lactic cultures determine the intensity of the flavor but that relative amounts are of great importance for the flavor quality. These investigators reported that an 21 acetaldehyde:diacetyl ratio of 1:4 existed in yogurt cultures of normal flavor. However, in a study by El-Sadec et a1. (1972) on zabady (the Egyptian fermented milk similar to yogurt) cultures, it was indicated that acetaldehyde is an important flavor component in zabady while the role of diacetyl appeared to be negligible. Although there have been various reports from many investigators on desirable acetaldehyde concentration in yogurt, on other carbonyl compounds and on their ratio, there has been increasing evidence that the presence of acetaldehyde is essential if the product is to have an acceptable flavor. A principal role in acetaldehyde production is attributed to L. bulcaricusJ although various strains of these species shown considerable differences in fermentation patterns. Hamdan et a1. (1971) examined the production of acetaldehyde by commercial yogurt cultures and their single strain components. A single strain of S. thermophilus, L: bulgaricus; and a 1:1 mixture of both were used separately to inoculate milk. L.bulgaricus produced more acetaldehyde (8 ppm) than S. thermophilus (4 ppm) during a period of five hr but the amount was still below that (25 ppm) formed when the organisms were grown together. From these results Hamdan et a1. (1971) not only supported the symbiotic relationship that exists between the two 22 organisms as indicated by other workers (Pette and Lolkema, 1950 a and b; Bautista et al., 1966; Galesloot et al., 1968; Veringa et al., 1968), but also demonstrated that L. bulgaricus is the principal organism in acetaldehyde .production in a yogurt culture. Cooling and Storage Cooling of yogurt serves to restrict further acid development. Mocquot and Hurel (1970) reported that cool- ing begins when the desirable pH of 4.5-4.7 is reached. Yeager (1975) suggested that cooling should start at pH 4.4-4.8 in order to obtain a final pH of 3.9-4.2. Kos- ikowski (1977) and Cottenie (1978) recommended pH 4.4 as a starting point for cooling. Rasic and Kurmann (1978) considered that pH 4.4 was too low. Stocklin (1969) claimed that the appearance of traces of whey on top of the coagulated yogurt is a better criterion to determine when to cool than pH or titratable acidity. Sellars (1973) and Tamime and Greig (1979) suggested that at the end of incubation the yogurt should be cooled as quickly as possible. According to Rasic and Kurmann (1978) the rate of cooling should not proceed too slowly or too fast since too slow cooling may result in additional increase in acidity and cooling too rapidly causes the formation of condensate and contraction of the gel. Kosikowski (1977) noted that a good forced-air blower system sufficient to chill the product within one hr or 23 less after entering into cold room is a major requirement in cooling. Cottenie (1978) described a cooling tunnel equipped with a conveyer and a cold-air blowing system at both sides. Most investigators agree that yogurt should be stored at or below 41°F to prevent over-acidification and micro- bial or enzymatic spoilage of yogurt. Kosikowski (1977), Kroger (1973) and Davis (1971) advocated the storage of yogurt at 40°F or lower. Humphreys and Plunkett (1969) recommended a temperature range of 39—410F for yogurt storage. Higher temperatures can lead to defects such as bitterness and low temperature to partial freezing and de- stabilization. Yogurt Drink The tremendous increase in yogurt consumption in the U.S. and all over the world has made food manufacturers look to other yogurt based dairy products as an opportuni- ty for product expansion. Yogurt drink has been introduced as one of the yogurt based dairy varieties which also in- clude yogurt salad dressing and yogurt mayonnaise. Yogurt drink is not a new concept, but earlier efforts to introduce this new product were unsuccessful because the market was not ready (Morley, 1979). The recent trend to acceptance of more varied yogurt products and the existing vast beverage market suggest a bright future for this liquid yogurt product. 24 Morley (1979) reported that about 10 dairy plants in the U.S. were manufacturing liquid yogurt drinks. Another report (Griffin, 1979) listed only eight dairy companies marketing yogurt drinks. Some stabilizer companies (Anonymous, 1978 a and b; Anonymous, 1979) introduced processing methods of yogurt drinks using proprietary blended stabilizers. Processigg Yogurt drinks currently produced can be classed into two somewhat arbitrary categories. One is the viscous and semiliquid type which is a heavy fluid but still drinkable. Morley (1979) defined this type of yogurt drink as a sweetened, low fat milk which has been cultured with the usual yogurt producing bacteria 8. thermophilus and L. bulgaricus and which is drinkable at refrigerator temperature. The other is a less viscous liquid drink made by diluting plain yogurt with water or aqueous flavorings. Flavoring materials (usually fruit), sweeteners and sta- bilizers are used in either type of drink. Duitschaever and Ketcheson (1974) studied the pro- duction of yogurt drink. Homogenized 2% fat milk was heated to 176°F for 20 min, cooled to 110°F and inocu- lated at this temperature with 3% yogurt culture and incubated to a pH 4.4-4.3. The coagulum was homogenized at 0-50 psi and heated to 128°F. Sugar, 0.25% high 25 methoxyl pectin and 0.25% ascorbic acid were added at this stage. Sugar (7-10%) was used when the flavor exe tract was used at a concentration of 0.1-1%, or 3-5% sugar was employed when natural flavor concentrates were used at concentrations of 5-10%. The product was then cooled to 60-68°F, homogenized at 2500 psi, packaged and stored at 39°F. Data obtained in taste panel evaluation showed good acceptance of natural orange and cherry flavored yo— gurt drinks. One commercial report (Anonymous, 1978 al described the procedure for making lowfat yogurt drink. The for- mulation suggested for this product is 0.5—2.0% fat, 8.25- 10.0% SNF, 0.2-0.3% stabilizer and 4.0-10.0% sugar. Fresh milk is standardized to the desired butterfat and SNF level with Grade A NFDM. Stabilizer is blended with sugar and added to the milk. The mixture is homogenized at 3,000 psi single stage after the temperature reaches 145°F, and heated to 1800F and held for 30 min. A frozen yogurt culture is added after the mix is cooled to lOOOF. When the yogurt reaches pH 4.3, it is cooled with slow agita— tion to 90°F, flavored and then cooled rapidly to 40°F. The product is held in 38°F cooler overnight to develop the desired body, texture and mothfeel. Morley (1978) proposed a procedure for manufacturing yogurt drink. Liquid milk products, NFDM, sugar and com— mercial yogurt drink stabilizer are blended. This mixture 26 is pasteurized at 180°F for 30 min, homogenized at the pasteurization temperature at 1300 psi, cooled to 108- llZoF, and then inoculated with a yogurt starter and agitated for 30 min. Incubation is continued without ag- itation to a pH of 4.3 over about 4-6 hr. The product is then agitated again and cooled to 45-500F. Flavors are added at about 10% level to the product with the recom- mended formula of 1.00% fat, 9.25% SNF, 5.50% sugar and 0.25% stabilizer. Another commercial report (Anonymous, 1979) suggested a method of manufacturing yOgurt drink using a special proprietary blended stabilizer. Liquid milk products, sugar and stabilizer are blended. This mixture is pas— teurized at 180-1900F for 30 min, homogenized at 1500-2000 psi, cooled to llO-llzoF and then inoculated with a yogurt starter. The product is incubated to a pH of 4.3 during 4-6 hr, cooled to 45°F and packaged. The proposed com— position of the product is 1.0% fat, 9.0% SNF, 5.0—6.0% sugar, and 0.1-0.2% stabilizer. Hekmati and Lame (1974) studied the production of a fermented milk product, known as "dough" which is widely consumed as a refreshing beverage in the Middle East areas. Yogurt was prepared from milk of 2.5% fat content and di- luted with an equal volume of water. Salt and flavoring essences were added. The mixture was homogenized at 2500 psi, bottled and held at low temperature. Sediment was 27 retarded by homogenization and low storage temperature. Composition of the product was 1.22% fat, 1.52% protein, 5.60% total solids, 1.0% salt and 0.006% flavoring essence. Another commercial report (Anonymous, 1978 b) de- scribed a method of producing fruit yogurt drink. A com- mercial proprietary stabilizer is dry blended with sugar and color. Another stabilizer solution (10% concentration) is stirred into yogurt. The dry blended mixture and flavor are added to the yogurt using a high speed stirrer. The pH is checked and adjusted to 4.0 to 4.1. The product is pasteurized at 1850F, homogenized at 1400 psi at the same temperature and hot (185°F) filled. The suggested compo- sition of the product is 75-85% skim milk yogurt, l4-24% fruit preparation, sugar, water and 1% stabilizer. Future of Yogurt Drink Yogurt drink is the combination of the natural in- gredients of yogurt with a refreshing taste of a liquid drink. Dairy companies currently producing yogurt drink report a favorable acceptance by consumers. Flavor and fruit suppliers have indicated that dairy processors and major beverage companies are moving quickly into this new product area (Griffin, 1979). Drucker (1973), Steinitz (1975), Morley (1978, 1979), and Griffin (1979) indicate that the prospects for yogurt drink are promising. Their predictions are based upon the 28 strong appeal of yogurt drink to dairy processors due to its versatility and relative ease of production, good con- sumer acceptance of it as a viable alternative to regular milks and soft drinks, and upon the huge beverage market which appears to be receptive to yogurt drink. However, Morley (1979) stresses the importance of producing a con- sistently good product and of creating a demand through creative advertising for this new product to be successful in the beverage market. MATERIALS AND METHODSa Preparation of Samples Preparation of Mother Culture Culture - A European SKI yogurt culture with slime forming capabilities was obtained from Michigan State Uni- versity dairy plant. Culture Medium - Spray-dried NFDM (Michigan Milk Pro- ducers Association, Ovid, Michigan) was reconstituted to 10% (W/W) as a medium for the propagation of culture. This reconstituted skim milk solution was dispensed into glass bottles (300 m1 and 100 ml), sterilized in the autoclave at 121°C for 10 min and stored in a refrigerator. Devices for Transfers - Pipets were dry sterilized at 180°C for 2 hrs. A pipet case was used for protection during sterilizing and storing pipets. Culture Room - A hood in the laboratory was used. The table inside the hood was wiped off thoroughly before use with 95% ethyl alcohol. Incubation - One percent culture was transferred to skim milk (10% solids) medium and the inoculated medium was held 45°C bath for 3 hr until coagulated. The culture was then cooled to 3-SOC and held in a refrigerator until the next propagation or use. Culture transfers were made every 5-7 days to assure proper activity. aMention of certain products or equipment does not imply endorsement. 29 30 Preparation of Yogurt Treatment of Milk - The milk used in this study was obtained from the Michigan State University dairy plant. Fat content of the milk was determined by Babcock method (AOAC, 1975). The milk was standardized to 1.5% fat and 12.0% NFS with spray-dried NFDM and water. The mix was pasteurized by a batch process at 88°C for 40 min in 10 gallon stainless steel milk cans, cooled rapidly to 60°C in an ice water bath and homogenized in a two stage Gaulin BW-40 homogenizer at pressures of 70 and 35 kg/cm2 on the first and second stages respectively. Incubation — The pasteurized and homogenized mix was cooled to 43-45°C and inoculated with 1.5% culture. The inoculated warm mixture was dispensed into glass beakers (1500 ml and 2000 m1) and incubated at 43-45°c until pH 4.4 was attained. The time required to reach this pH was usually about 3 hr. Cooling and Cold Storage - The yogurt was then cooled to 3°C in a moving-air cold room and maintained at this temperature for subsequent use in the preparation of yogurt drink. The procedures for yogurt preparation are outlined schematically in Figure 1. Preparation of Yogurt Drink Composition - Yogurt drink consisted of 25% low fat yogurt previously prepared, 10% frozen concentrated orange juice (4+1), 9% sugar and 56% water. The frozen concentrated 31 lStandardization of milfi l Pasteurization I Homogenization | Cooling Culture preparation ———+ Inoculation I Incubation I Cooling | Cold storage Fat: 1.5% NFS: 12.0% 88°C for 40 min 60°C First stage: 70 kg/cm Second sta e: 35 kg/cm 43-45°c 1.5% inoculum 43-45°c 3 C 3 C Figure 1. Flow diagram of yogurt manufacture. 32 orange juice was obtained from the Michigan State,Univer- sity food stores. Optimal sugar level was determined by sensory evaluation using ranking method (see sensory evaluation). Processing - The ingredients were blended with a high speed mixer for 3 min in 5 or 10 gallon milk cans and dispensed in equal volume (5.0 liters) into large stain- less beakers for processing to evaluate the experimental variables of pH, pasteurization temperature/time, homo- genization pressure and stabilizers (see experimental variables). The pH of the mixture was adjusted using 50% food grade lactic acid (Monsanto Co., St. Louis, Missouri) and the stabilizers were added at this stage. The mixture was pasteurized either by a batch process or by means of a Cherry Burrell (UHT) heat exchanger (Spirotherml equipped with a spiral holding tube, cooled to 60°C in an ice water bath and then homogenized in a two stage Gaulin BW-40 homogenizer. Cooling_and Cold Storage - The homogenized product was cooled rapidly to 5°C in an ice water bath and stored at 5°C. The procedures for yogurt drink preparation are out- lined in Figure 2. Determination of Fat and Total Solids in Yogurt and Yogurt Drink Percentage of fat and total solids of yogurt and yo- gurt drink were determined by the Mojonnier modification of 33 Low fat yogurt Water Orange juice Sugafi "1"— I I I I I I I Stabilizefi I Blending pH control Lactic acid I Pasteurization Batch process or UHT I Homogenization I 60°C I Cooling I Ice water bath, I l Cold storage I 5°C 5 C Figure 2. Flow diagram of yogurt drink manufacture. 34 the Roese-Gottlieb extraction procedure and the Mojonnier vacuum oven procedure (Mojonnier and Troy, 1972) respectively. EXperimental Variables in the Preparation of Yogurt Drink Samples The effect of pH, pasteurization temperature/time, homogenization pressure and stabilizers on the stability and sensory properties of yogurt drink was studied. ProcessingiParameters BE — Acidity of yogurt drink samples was adjusted to four pH levels using 50% food grade lactic acid; 3.2, 3.6, 4.0, and 4.2. Pasteurization Temperature/Time - Yogurt drink samples were pasteurized at four pasteurization temperature/time combinations: 65°C for 30 min, 75°C for 10 min, 88°C for 2 min (batch process), and 115°C for 4 sec (UHT). Homogenization Pressure - Yogurt drink samples were homogenized at four different pressures at 60°C; 70 kg/cmz. 105 kg/cmz, 140 kg/cm2 and 175 kg/cmz, with second stage con- stant at 35 kg/cm2 and first stage increasing by 35 kg/cm2 from 35 kg/cm2 to 140 kg/cmz. Stabilizers Guar gum carrageenan, pectin, propylene glycol al- ginate were used individually at four concentration levels: 0.05, 0.10, 0.15 and 0.20%. 35 Carboxymethyl cellulose gum (CMC) was used in combin- ation with guar gum as follows: 1) Guar gum 0.20% + CMC 0.05% 2) Guar gum 0.15% + CMC 0.05% 3) Guar gum 0.15% + CMC 0.10% 4) Guar gum 0.00% + CMC 0.15% Guar gum.was also evaluated in combination with propylene glycol alginate and pectin: l) Guar gum 0.10% + propylene glycol alginate 0.10% 2) Guar gum 0.10% + pectin 0.10% One commercial proprietary yogurt drink stabilizer composed of gelatin, locust bean gum and carrageenan was evaluated at six concentration levels: 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30%. Stability Studies Sediment Sedimentation was observed in the yogurt drink samples (see determination of optimum processing parameters).to study the effect of processing parameters on the stability of the product. The samples after cooling were dispensed into 250 ml graduated cylinders and stored in a refrigerator at 5°C. The amount of sediment was measured after storage for 5 and 8 days. 36 Syneresis If syneresis was observed in the yogurt drink samples, it was noted and recorded to indicate the effect of sta- bilizers on the stability of the product. The samples after cooling were dispensed into 250 ml graduated cylinders and stored in a refrigerator at 5°C. The amount of syneresis was checked 3, 5, 8 and 10 days after storage. Protein determination The supernatant fluid of yogurt drink samples was de- canted into small beakers immediately after final checking of sediment or syneresis and protein content was determined on them. Total nitrogen was determined using a semimicro Kjeldahl method (Swaisgood, 1963; Harte, 1978). Samples containing approximately 15 mg of nitrogen were digested with 5 m1 of digestion mixture on an electric burner for 90 min. The digestion mixture consisted of 5 g SeO2 and 5 g CuSgySHZO in 500 ml of concentrated H2504. After the initial digestion, the samples were cooled, 2 ml of 30% H202 was added and digestion continued for another 90 min. After cooling, the flasks were rinsed down with 10 m1 of redistilled water. Immediately after the flasks were attached to a steam distillation apparatus, approximately 25 m1 of 40% NaOH was added. The released ammonia was steam distilled and collected into 15 ml of 4% boric acid solution containing 5 drops of mixed indicator. The 37 indicator consisted of 400 mg bromocresol green and 40 mg methyl red in 100 ml of 95% ethanol. Standard 0.0205 N HCl was used to titrate the sample. The nitrogen deter- mination was performed in duplicate and total nitrogen values were converted to crude protein using a factor of 6.38. The recovery of nitrogen from standards was 98.11 i 1.26% using L-tryptophan. SensoryiEvaluation In this study sensory subjective analysis was carried out to determine the sugar content of yogurt drink and to determine the effect of processing parameters and stabil- izers on the sensory properties of yogurt drink. Two types of preference test were employed for this purpose: rank— ing method and hedonic scaling method (Amerine et al., 1965; Lavery, 1972; Larmond, 1977). Ranking Method Ranking method was used to determine the sugar con- tent of yogurt drink and the effect of homogenization pressure on the sensory properties of the product. In this method the panelists were presented with randomly coded samples to rank in order of preference. To deter- mine if the difference between the sample is significant at the 5% level of significance, the rank totals were compared with the values in Table 23 of the Appendix. When one or more rank sums were lower than the upper left 38 value in the block and higher than the upper left value, statistical significance was indicated. Hedonic Scaling Method A nine-point hedonic scaling method were used to de- termine the effect of pH, pasteurization temperature/time and stabilizers on the sensory properties of the product. In this method the panelist were presented with randomly coded samples to express their degree of liking or dis- liking. The ratings for each sample were given numerical values ranging from like extremely (9) to dislike extremely (l). The results were anlyzed by analysis of variance, for which the correction factor (CF), the sum of squares ($3), the degrees of freedom (df), the mean square (ms), and the variance ratio (F) were calculated. To determine if the difference between the samples is significant at the 5% level of significance, the calculated P value was checked on Table 24 of the Appendix. In this method, the variance ratio (F) must exceed tabular value to be significant. When there was a significant difference between the samples, those which were different were determined using Tukey's test (Snedecor, 1956). Least significant difference was calculated and any two sample means (score/number of judge- ments) that differ by the value of least significant dif- ference or more were considered to be different significantly. 39 Determination of Optimum ProcessinggParameters Yogurt drink mix was subjected to various treatments (combinations of processing parameters) in order to de- termine optimum pH, pasteurization temperature/time and homogenization pressure on the stability and sensory properties of the product. All runs were made in dupli- cate. P_H_ The experimental runs are shown in Table 4. The runs from No. 1 to No. 6 were used for sensory evaluation and the runs from No. 7 to No. 14 for sediment observation and protein determination. Pasteurization Temperature/Time The runs from No. 7 to No. 14 in Table 4 were used for sediment observation and protein determination. The runs made for sensory evaluation are shown in Table 5. Homogenization Pressure The runs made are shown in Table 6. All runs were used for sediment observation and protein determination, and runs from No. 1 to No. 4 for sensory evaluation. 40 Table 4. Treatments of yogurt drink to determine optimum pH on the stability and sensory properties of the product. Pasteurization Run pH temperature/ Homogenization Remarks No. time pressure 1 3.6 65°C/30 min 140 kg/cm2 Sensory evaluation 2 4.0 65°C/30 min 140 kg/cm2 Sensory evaluation 3 4.2 65°C/30 min 140 kg/cm2 - Sensory evaluation 4 3.6 115°C/4 sec 140 kg/cm2 Sensory evaluation 5 4.0 115°C/4 sec 140 kg/cm2 Sensory evaluation 6 4.2 115°C/4 sec 140 kg/cm2 Sensory evaluation a7(2) 4.0 65°C/30 min 140 kg/cm2 Sensory evaluation a8(3) 4.2 65°C/3O min 140 kg/cm2 Sediment, protein de- termination 9 4.0 75°C/10 min 140 kg/cm2 Sediment, protein de- termination 10 4.2 75°C/10 min 140 kg/cm2 Sediment, protein de- termination 11 4.0 88°C/2 min 140 kg/cm2 Sediment, protein de- termination 12 4.2 88°C/2 min 140 kg/cm2 Sediment, protein de- termination a13(5) 4.0 115°C/4 sec 140 kg/cm2 Sediment, protein de- termination a14(6) 4.2 115°C/4 sec 140 kg/cm2 Sediment, protein de- termination a TWO runs are same treatments. Table 5. Treatmentsa of yogurt drink to determine 41 optimum pasteurization temperature/time on the stability and sensory properties of the product. Pasteurization Run pH temperature Homogenization Remarks No. time pressure 1 4.0 65°C/30 min 140 kg/cm2 Sensory evaluation 0 2 4.0 75 C/10 min 140 kg/cm2 Sensory evaluation 3 4.0 88°C/ 2 min 140 kg/cm2 Sensory evaluation 4 4.0 115°C/ 4 sec 140 kg/cm2 Sensory evaluation aThe treatments from No. 7 to No. 14 in Table 4 were used for sediment observation and protein determination. 4 2 Table 6. Treatments of yogurt drink to determine optimum homogenization pressure on the stability and sensory properties of the product. Run pH No. Pasteurization temperature/ time Homogenization Remarks pressure 88°C/ 2 88°C/ 2 -88°C/ 2 88°C/ 2 115°C/ 4 115°C/ 4 115°C/ 4 115°C/ 4 min min min min SEC sec sec SEC 70 kg/cm2 Sensory evaluation, sediment, protein de- 2 termination 105 kg/cm Sensory evaluation sediment, protein de- 2 termination 140 kg/cm Sensory evaluation, sediment, protein de- 2 termination 175 kg/cm Sensory evaluation, sediment, protein de- 2 termiation 70 kg/cm Sediment, protein de- 2 termination 105 kg/cm Sediment, protein de- 2 termination 140 kg/cm Sediment, protein de- 2 termination 175 kg/cm Sediment, protein de- termination 43 Determination of the Most Suitable Stabilizers and their Concentrations in Yogurt Drink Various stabilizers at different concentrations (see experimental variables) were tested for their stabilizing effects at the predetermined processing parameters. Sta- bilizers were premixed with sugar and then the dry mixture was added to the mixture of yogurt and water under vig- orous agitation until a uniform suspension was obtained. Stability studies were conducted on every sample made by observation of syneresis and protein determination. Sen- sory evaluation was carried out only on the two samples finally stabilized. RESULTS AND DI SCUSS ION Composition of Yogurt and Yogurt Drink Mean values plus or minus standard deviation for the content of protein (Kjeldahl), fat (Mojonnier) and total solids (Mojonnier) of yogurt and yogurt drink are shown in Table 7. The composition of low fat yogurt made for this study was carefully controlled by precise stan- dardization of ingredients since commercial yogurts, as previously mentioned, are subject to compositional var- iables which lead to undesirable complications regarding the analysis of experimental data. The approximate ratio of one (yogurt) to two (water) in yogurt drink was chosen for optimal viscosity and mouthfeel. Frozen concentrated orange juice was selected as natural flavor ingredient and was used at a concentration of 10%. At concentrations of 20 and 15%, the orange flavor was too strong and the unique flavor of yogurt was masked. In Table 7, the values of protein and fat content in yogurt drink are higher than those of the same constitu- ents divided by four (25% of yogurt was used in yogurt drink) in yogurt, due to the composition of the orange juice. According to data of the U.S. Department of Ag- riculture (1975), frozen concentrated orange juice contains 2.5% protein, 0.2% fat and 44.8% total solids. 2 The desired sugar level of yogurt drink was deter- mined by subjective sensory evaluation using ranking method. 44 45 Table 7. Chemical composition of yogurt and yogurt drink.a Product Proteinb FatD Total solidsb Yogurt 4.67:0.02 1.54:0.03 l3.63r0.04 Yogurt drink 1.43:0.02 0.41:0.02 17.12r0.06 aVariation indicated by standard deviation of duplicate banalysis from two runs. % , w/w Sugar concentrations of 7%, 8%, 9% and 10% were tested. Sugar concentrations of 4%, 5% and 6% were eliminated during preliminary evaluations because the yogurt drink was too sour. The panelists were presented with four randomly coded samples (each containing different sugar levels) to rank in order of preference. The rank scores given to the samples by the judges are shown in Table 8. A low score indicates greater acceptability (preference) in this method. The rank totals were compared with the values in Table 23 of the Appendix. Since there were four treatments and eight judges, the tabular entries are 13-27. The lowest insignificant rank sum is 13 and the highest insignificant rank sum is 27. One rank sum (11) was lower than 13, three other rank sums were between the range of 13-27 and no rank sum was higher than 27. To show significant difference statistically, one or more rank sums should be lower than 13 and higher than 27. There was no significant difference in preference for the samples at 5% level of significance. However, the lowest 46 Table 8. Ranked scores of yogurt drink at the sugar concentrations of 7%, 8%, 9% and 10%. Judges Sugar level 7% 8% 9% 10% l 4 3 l 2 2 4 3 l 2 3 2 4 l 3 4 3 l 2 4 5 4 3 l 2 6 l 2 3 4 7 4 2 l 3 8 3 2 l 4 Total 25 20 11 24 47 rank sum (11) of the sample treated with 9% sugar indi- cated apparent preference for this sample to the other three samples. The judges who showed less preference for the sample treated with 10% sugar indicated that the pro- duct was too sweet and those who showed less preference for the samples with 7% and 8% sugar commented that both samples were too sour. Basing on this analysis a sugar concentration of 9% was selected for use in the yogurt drinks. Chemical and Physical Properties of Yogurt Drink In order to study the effects of various processing parameters and stabilizers on the stability of yogurt drink, it is reasonable to define the chemical and physical nature of this particular product. The yogurt drink used in this study was composed of 25% yogurt, 10% concentrated orange juice (4 + l), 9% sugar and 56% water. The constituents of each component which contribute to the characteristics of the yogurt drink are milk proteins (caseins and whey), milk fat, lactose and salts from the yogurt; small amounts of proteins, lipids (orange oil), carbohydrates and trace amounts of salts from orange juice; the sucrose solids. Milk proteins do exist not in their original form, but in changed form due to the treatments during the manufacture of yogurt. The casein originally is present in milk in the form of small micelles or in smaller 48 polymeric units and monomeric forms, comprising from 5-10% of the total casein. The micelles referred to as a calcium caseinate-phosphate-complex contain, in addition to casein, inorganic calcium and phosphate, a little magnesium and citrate. Casein is not a heat-coagulable protein in the strictest sense. In normal fluid milk casein is very stable to heat. Parry (1974) stated that casein resists coagulation for as long as 14 hr at boiling temperature and 1 hr at 130°C. During the manufacture of yogurt, however, there is a gradual removal of calcium and phosphorus from the colloidal complex to soluble state due to the acid produced by the yogurt culture. At pH 5.2-5.3 the casein particles are destabilized initiating precipitation. Complete precipitation occurs at pH 4.6-4.7, the isoelectric point, with the casein existing in a salt-free state. In contrast to casein, whey proteins in the native state are not sensitive to changes in hydrogen ion concentration. They are affected considerably by heat treatment of the milk. Above the normal pasteurization temperature of milk, the whey proteins are denatured and their Specific structures are disrupted and unfolded. Lar- son and Rolleri (1955) reported that almost complete de- naturation of whey protein occurred by heating milk at 77.5°C for 1 hr, 80°C for 30 min or 90°C for 5 min. In the present study, the yogurt mix was pasteurized at 88°C for 40 min. It is apparent that essentially all whey proteins in the yogurt used for yogurt drink had been denatured. 49 During the fermentation of yogurt, usually 20-30% of the lactose is fermented into lactic acid as previously mentioned. Calcium removed from the casein micelles mostly combines with lactic acid as calcium lactate. Fat globules in milk are finely divided and mechanically incorporated within the coagulum structure by homogeniza- tion during the manufacture of yogurt. Thus, the semi- solid gel structure of yogurt represent a co-precipitate of destabilized casein and denatured whey proteins entrap- ping the fat globules and serum with dissolved constituents. When all ingredients are blended using a high speed mixer for the manufacture of yogurt drink, the gel structure of yogurt is completely broken and consequently the agglo- meration of casein micelles associated with denatured whey proteins may exist in the form of a micro-dispersion, while a part of the casein and undenatured proteose-peptone fraction are in true colloidal dispersion and the non- protein nitrogen compounds such as amino acids and urea, lactose and free salts are in true solubilized solution. The milk fat globules would exist as an oil-in-water emul- sion in the mixture, and the sucrose would be completely dissolved in the system. The proteins of the orange juice are likewise dispersed colloidally, the orange lipids as an emulsion and the carbohydrates of the orange juice will be in true solution in the aqueous phase of the yogurt drink. 50 Effect of Processipg Parameters on the Stability and Sensory Properties of Yogurt Drink In this study, the first priority was given to the results of sensory evaluation for choosing optimum pro- cessing parameters since food products should be appealing and palatable. Therefore, sensory tests were performed prior to stability studies and when sensory evaluation data indicated that a significant difference existed be- tween (among) the samples, only those treatments which gave satisfactory results were employed in subsequent ex- periments. Regarding the stability of this system, the milk proteins are the constituents most sensitive to pH, heat treatment and homogenization pressure. It should be noted here that the caseins of yogurt and the denatured whey pro- teins which adsorb or associate with them, are of primary importance to the body and rheological prOperties of the yogurt drink. Therefore, protein determination on the supernatant fluid of yogurt drink as well as measurement of sediment were employed to measure stability. pg Sensory evaluation was performed to find out the effect of pH on the sensory properties of yogurt drink. The processing parameters employed for this sensory analysis are shown in Table 4. pH 3.2 was excluded in this sensory 51 evaluation since this concentration of H+ imparted an extremely sour flavor to the product in the preliminary evaluation by a small group of panelists. One heat treat- ment (65°C/30 min) was chosen from the three conventional methods employed in this study and the other was UHT (115°C/4 sec). A nine point hedonic scaling method which is most commonly used for preference testing was used. The results are shown in Table 9 and an analysis of vari- ance was conducted on the data of Table 9, as shown in Table 10. The calculated variance ratio P value (5.73) was checked on Table 24 of the Appendix with 5 df in the numerator and 105 df in the denominator. The variance ratio F value (5.73) exceeded the tabular value (2.29), thus the difference between samples was indicated as sig- nificant. To determine the samples that are significantly different, least significant difference (1.35) was calcu- lated by Tukey's test. The differences of mean values are presented in Table 11. For any two samples to be significantly different, the difference of their mean values should be greater than least significant difference (1.35). As shown in Table 11, the differences between the samples that are greater than 1.35 are from B-(A or D) and E-(A or D). Therefore, the samples B and E are sig- nificantly preferred to the samples A or D. It can be also found that the samples B and E are preferred much more if not significant to the samples C or F. The 52 mucmEomppn mo Honspc\muoomp oupmmoum componwcomosomo oEflu\ousumummEou compenwusoummmn mom me.m mm.o no.4 me.m Ne.o ma.e memos onSmmo Hme oNH see mos oNH mes Noe Hence em o o o a e o NN AN N m e H o e NN NN m m N e e N oN mm A m o N e e as -He o a e m e e we oN m e m e e N as em N e o N o o as NN e e m N e m me mm m a o e o a ea NN m e m m m m NH em o o o e o e NN Hm m m e m m m as em a e o e e m as Nm o o e e e m a Na N o o a e o N Ne w m o e e o e em a e e m e e r em N o m e e N m NN e m m m o o a an m a m m e o m me e e N m N a N as m N m e o m H Naoxoxoee Nao\osoeN Nao\oxoeH Naoxoxoee Nao\mxoee Nao\oxoeeo Hmooe sexoomee =e\oomHH =e\oomHN .omxoomo .omxoomo .om\oomo o nooose N.e o.e e.m N.e o.e o.m m o m o o m a mflcwfifimmufi .xcHHp pupmo> mo moan Inwmonm muomcmm one :0 mm mo muoowmm on» mppum on com: mmHmEmm 05» mo mmhoom .m manna Table 10. 53 Analysis of variance from the data of Table 9. Source of variation df F Samples 5 68.13 a5.73 Judges 21 112.10 2.24 Error 105 250.04 asignificant at 0.05 Table 11. Difference of sample means from data of Table 9. Treatments Mean values Difference of mean values B-°(A or 0) 6.77 4.95 1.82 B- (C or F) 6.77 5.45 1.32 B-E 6.77 6.55 0.22 E- (A or D) 6.55 4.95 1.60 E- (C or F) 6.55 5.45 1.10 (C or F) - (A or D) 5.45 4.95 0.50 a( ) - Mean values of both samples are same. 54 conclusion derived from the Tables 9, 10 and 11 is that the samples adjusted to pH 4.0 were significantly preferred to the samples adjusted to pH 3.6 and much more preferred to the samples adjusted to pH 4.2 at the 5% level of signifi- cance. So pH 4.0 was taken as the best for the product and pH 3.6 was excluded in the subsequent experiments. The samples adjusted to pH 3.6 were described as too sour. As for the samples adjusted to pH 4.2, many panelists evaluated them as a little too sweet. This also supports the result of preliminary evaluation with a smaller group of panelists. In the preliminary evaluation, products with a pH of 3.2 had been rated as very sour flavor, so this. pH was not used in the sensory evaluations. The influence of pH on the sediment volume of the samples 5 and 8 days after storage are shown in Table 12. The corresponding Kjeldahl protein data on the super- natant fluid of the samples 8 days after storage are shown in Table 13. In this experiment, pH variation was nar- rowed from pH 3.2, 3.6, 4.0 and 4.2 to pH 4.0 and 4.2 based on the results of previous sensory evaluation. Four different heat treatments were used (the data from this trial were also to be used for studying the effect of heat treatments on the product stability) at a constant homogenization pressure, 140 kg/cmz. From Table 12 and 13, it is evident that the increase in sediment volume with increase in pH of the product at all heat treatments 55 Table 12. Sediment volume of the samples used to study the effect of pH on the stability of yogurt drink. Treatments ‘gaSediment (ml) pH Pasteurization Homogenization 5 days 8 days temperature/time pressure 4.0 65°C/30 min 140 kg/cm2 219 196 4.2 65°C/30 min 140 kg/cm2 222 200 4.0 75°C/10 min 140 kg/cm2 215 192 4.2 75°C/10 min 140 kg/cm2 .218 197 4.0 88°C/2 min 140 kg/cm2 160 126 4.2 88°C/2 min 140 kg/cm2 171 129 4.0 115°C/4 sec 140 kg/cm2 35 34 4.2 115°C/4 sec 140 kg/cm2 37 35 aAverage of duplicate runs. 56 Table 13. Protein content of supernatant fluid of the samples used to study the effect of pH on the stability of yogurt drink. Treatments aProtein (%) pH Pasteurization HomogeniEation ~ temperature/time pressure 4.0 65°C/30 min 140 kg/cm2 0.37 4.2 65°C/30 min 140 kg/cm2 0.33 4.0 75°C/10 min 140 kg/cm2 0.41 4.2 75°C/10 min 140 kg/cm2 0.35 4.0 88°C/2 min 140 kg/cm: 0.44 4.2 88°C/2 min 140 kg/cm 0.41 4.0 115°C/4 sec 140 kg/em2 0.91 4.2 115°C/4 sec 140 kg/cm2 0.59 aAverage of duplicates from two runs. 57 used is responsible for the decrease of protein content in the supernatant fluid of the product. The decrease of sediment volume in each sample during prolonged storage is viewed as resulting mainly from the contraction of sediment portion. A Slight reduction in the pH of yogurt drink resulted in a noticable decrease of sedimentation and increase of the stable protein in the supernatant. From these results, it appears that some control of sed- iment formation can be achieved by adjusting pH of the product to 4.0. Based on the fact that the isoelectric point of casein is 4.6, it seems possible that pH 4.2, being closer to the isoelectric point of casein than pH 4.0 is, caused more sedimentation and less stable protein in the supernatant. The solubility of casein is minimum at the isoelectric point and increases rapidly on either side. Pasteurization Temperature/Time Sensory evaluation was conducted to determine the effect of pasteurization temperature/time on the sensory properties of yogurt drink. The processing parameters used in this sensory analysis are shown in Table 5. pH 4.0 was used since it was proven in the previous investi- gations to be the best for the stability and sensory properties of the product. All of the four heat treat- ments were used with a homogenization pressure constant at 140 kg/cmz. 58 oupmmmum coauMchomOEomo meflu\musumuomEmu compouflupoummm new mom wed mmH wvH mmH kuoa mm m m h o mm em m m m o Hm mm m n m m cm mm v e m n ma mm m h h n ma an m m m m ma mm m m h n ma mm m m m n ma om m e w m «H mm m o m m ma em m h m n «H mm m m m m AH mm m m m b ca cm s e o m m mm m e m e m cm e o m m m mm m m m m m mm o m m m m mm m m w e V cm m m m m m Hm m m m m N mm m m m m H Nao\ox oeH Nso\ms oee Nao\ox oeH Nao\mx oeeo Hmuoe svmwOmHH .NWUOmm .om\00m> .om\00mmn mompph e o e o e c on mucoEumone .xcwnp pupmom mo mmwuummoum whomcom msu so ofiwu\oupumummfimu coflumuflupoumwm mo muomwmm one appum on com: mmHmEmm may mo mouoom .eH canoe 59 Table 15. Analysis of variance from the data of Table 14. Source of df 33 ms F variation Samples 3 4.44 2.22 a1.46 Judges 21 61.19 2.91 1.91 Error 63 95.81 1.52 aSignificant at 5% level. A nine point hedonic scaling method was also used in this sensory evaluation. The scores of each sample given by 22 panelists are presented in Table 14. Analysis of variance was conducted on the data of Table 14, as shown in Table 15.. The calculated variance ratio F value was checked on Table 24 of the Appendix with 3 df in the numer- ator and 63 df in the denominator. The variance ratio (1.46) was smaller than the tabular value (2.76). So there was no signficant difference in preference for the samples at the 5% level of significance. This indicates that heat treatment variation had negligible effect on the sensory properties of the product. Data presented in Table 12 and 13 were also used to elucidate the effect of heat treatment variation on the stability of yogurt drink. Sediment volume decreased con- siderably with increasing temperature at the both pH 4.0 and 4.2 and corresponding increases of protein content in 60 the supernatant also occurred although there was little difference between the heat treatments of 65°C/30 min and 75°C/10 min. This difference may be due partially to the result of differing extents of B-lactoglobulin-k- casein interactions occurring during heating of the samples. The extent of this interaction is held to play an impor- tant role in the heat stability of milk system. The heat induced interaction of B-lactoglobulin and k-casein is believed to proceed through the mechanism of disulfide interchange involving the exposed thiol groups of B- lactoglobulin and the disulfides of k-casein. In yogurt drink, some interaction may have been already present as a result of heat treatment given during the manufacture of yogurt. More interactions may have been induced during the heat treatment of yogurt dirnk, the higher temperature resulting in the greater extent of interaction. Brunner (1977) stated that the association of B-lactoglobulin with k-casein increases the heat stability of colloidal casein. Changes in the ionic calcium content of the samples induced by heat treatment may be partially responsible for the different stability of the product. It has been reported that ionic calcium plays a role in the stability of milk system, higher calcium ion causing lowered heat stability and that heat treatment results in a transfer of calcium from the ionic to the colloidal state at the higher temperature heat treatment, resulting in the increased stability. 61 This result may be also attributable partially to the increased hydration of proteins in the system at the higher temperature. When in solution, proteins apparently are hydrated in the sense that a substantial amount of water is bound or trapped by the protein. Increased hy- dration if not excessive favors the stabilization of proteins. Homogenization Pressure Sensory evaluation was conducted to determine the effect of homogenization pressure on the sensory proper- ties of yogurt drink. The processing parameters employed in this sensory analysis are shown in Table 6. ZipH 4.0 was maintained for these experiments as this pH had proven best for the stability and sensory properties of the product. Only one heat treatment at 88°C/2 min was used since this temperature/time was optimal for the stability of product among those three conventional heat treatments tried in this study. Variation in heat treat- ment also apparently has negligible effect on the sensory properties of the product. Ranking method was used in this sensory evaluation. The panelists were presented with four randomly coded samples treated at four different homogenization pressures respectively with the same pH and heat treatment and asked to rank in order of preference. The ranks given to the samples by the panelists are 62 0H5 momma coeumecmmoEom mad .U\0H5UMHOQE$# COHHMNHHSTUMM . . & mm m a o 4 mm me man M m N Hmuoe m m e H H e e m mH N N H H NH m e m N oH m H m a mH N N N H 4H H H H 4 NH m e e 4 NH N N N m HH m m e m OH H N .a H a H m e H m e N e H e N m m N o H H N e m m . . W N m Nao\ox meH e N .N\oomm Naoxox oeH So H O.v .N\UOmm N \Ox mOH o.e .N\oome Nau\os oeo mfiGTEuMOHB o.v .NMWWmmfl 6 mmmpph coNumNHcmmo .xcHH . . Eon mo . p pupae» muommmo on» monumwo mmwuuomoum Shows on pomp moamfimm pawn may :0 onpmmmum mo mmuoom pmxcmm . ma oHnme 63 presented in Table 16. AS mentioned previously, low score indicates greater acceptability (preference) in this method. The rank totals were compared with the values in Table 23 of the Appendix. Since there were four treatments and eighteen judges, the tabular entries are 34-56. The lowest insignificant rank sum is 34 and the highest in- significant rank sum is 56. To show significant difference statistically, one or more rank sums whould be lower than 34 or higher than 56. All ranks sums in Table 16 are be- tween the range of 34-56. The conclusion is that there was no significant difference in preference for the samples evaluated. This result indicates that homogenization pressure variation had little effect on the sensory proper- ties of the product. The processing parameters used for the stability study are also shown in Table 6. pH was held constant at 4.0. Two heat treatments, 88°C/2 min from the three conventional methods and 115°C/4 sec (UHT), were used as heat treatment variation. Sediment volume 5 and 8 days after storage of the samples are shown in Table 17. The corresponding Kjeldahl protein data on the supernatant fluid of the samples 8 days after storage are presented in Table 18. Data in Table 1? reveal that a considerable in- crease in sediment volume occurred with increasing homo- genization pressure at either heat treatment, indicating that homogenization definitely had much effect on the 64 Table 17. Sediment volume of the samples used to study the effect of homogenization pressure on the stability of yogurt drink. Treatments aSediment (ml) pH Pasteurization Homogenization 5 days 8 days temperature/time pressure 4.0 88°C/2 min 70 kg/cm2 125 94 4.0 88°C/2 min 105 kg/cm2 143 112 4.0 88°C/2 min 140 kg/cm2 156 123 4.0 88°C/2 min 175 kg/cm2 159 ' 128 4.0 115°c/4 sec 70 kg/cm2 30 30 4.0 115°C/4 sec 105 kg/cm2 34 33 4.0 115°C/4 sec 140 kg/cm2 35 34 4.0 115°C/4 sec 175 kg/cm2 37 36 a I Average of duplicate runs 65 Table 18. Protein content of supernatant fluid of the samples used to study the effect of homogeni- zation pressure on the stability of yogurt drink. Treatments Pasteurization Homogenization aProtein (%) pH temperature/time pressure 0 . 2 4.0 88 C/2 min 70 kg/cm 0.66 4.0 88°C/2 min 105 kg/cm2 0.51 4.0 88°C/2 min 140 kg/cm2 0.44 4.0 88°C/2 min 175 kg/cm2 0.30 4.0 115°C/4 sec 70 kg/cm2 1.05 4.0 115°C/4 sec 105 kg/cm2 0.92 4.0 115°C/4 sec 140 kg/cm2 0.89 4.0 115°C/4 sec 175 kg/cm2 0.79 aAverage of duplicates from two runs. 66 stability of the product. A corresponding decrease in protein contents of the supernatant fluid of the product also occurred. Parry (1974) stated that nonionic and inert materials such as fats, sugars and fruit pulps appear to adsorb protein in milk systems, finer disper- sion tending to concentrate the protein with increasing effectiveness and this accumulation on the interface tends to lower protein stability by encouraging local coagulation, which quickly destabilizes the entire system. Homogenization increases the dispersion of these materi- als, resulting in more surface area for adsorption. In the present study, the lowered stability of the product .at the higher homogenization pressure is believed due to the finer dispersion of the fat and the pulp (from orange juice) and their increased surfaces for adsorption of proteins. The system of yogurt drink used in this study can be closely compared to the system of evaporated milk in the sense that proteins in both are heat treated previously and that both systems have milk fat. Hun- ziker (1949) and Newstead et a1. (1978) stated that homogenization tends to lower the stability of evaporated milk and this tendency increases with increasing homo- genization pressure due to the increased formation of fat-protein complex which decreases the stability of the system. 67 Effect of Stabilizers on the Stability and Sensory Properties of Yogurt Drink It has been shown in the previous investigations of this study that the optimum processing parameters for yogurt drink are pH 4.0, heat treatment 115°C/4 sec (UHT) and homogenization pressure 70 kg/cmz. In this study of stabilizers, however, 88°C/2 min rather than UHT was used for heat treatment considering that not all dairy plants have UHT facilities and 175 kg/cm2 rather than 70 kg/cm2 was used as homogenization pressure for better distribution of the stabilizers in the product. The stabilizers used in this study were expected to fulfill the following requirements: bland flavor (no masking of the flavorings), easy to incorporate, readily hydrated, stable, and imparting the desired stabilizing effects (no syneresis) at low concentration without im- parting excessive viscosity (heavy mouthfeel) to the product. Complete dissolution (no clumping) of the stabilizers was achieved successfully by premixing with sugar and adding the mix to the product under vigorous agitation. Concentration of each stabilizer was tried in the se- quential order from a low concentration (0.05%) to high, increasing by increments of 0.05%. When certain level of a stabilizer gave too much viscosity to the product, 68 higher concentrations of the stabilizer were not tried even if the product was still not stabilized. Syneresis volume of yogurt drink samples after storage for 3, 5, 8 and 10 days as a function of individual stabilizer at different concentrations are shown in Table 19-20 and plotted in Figure 3-7. The results of protein determination on the syneresis after storage for 10 days are also shown in Table 19 and 20. The stability characteristics of yogurt drink system are of course related to the suspension of hydrated colloids in a aqueous medium. Stabilizers in this type of system are expected to maintain particles in maximum state of suspension and take up free water as water of hydration through intra- and intermolecular interactions. Syneresis of the samples on standing was interpreted as evidence of instability. Carrageenan (Table 19, Figure 3) proved to be in- effective in stabilizing this system. During initial storage, the stability of the yogurt drink containing higher levels of carrageenan was improved. However, stability diminished markedly with time. After 10 days, almost identical volumes of syneresed fluid were observed in all samples. Increase of the concentration from 0.20% to 0.25% (the broken line) not only caused an adverse effect on the stability, but also imparted apparent vis- cosity to the product. 69 Table 19. aSyneresis volume (ml) of yogurt drink samples after storage as a function of stabilizers at different concentrations. pH:4.0, heat treatmenté 88°C/2 min, homogenization pressure: 175 kg/cm . Storage 0 Concen- (Days at 5 C) Remarks tration(%) 3 5 8 10 Protein (mouthfeel) Carrageenan 0.05 108 120 135 141 0.31 0.10 86 114 135 141 0.31 0.15 77 108 133 140 0.31 0.20 60 88 123 132 0.28 0.25 76 108 125 130 0.28 Heavy Guar gum 0.05 24 41 76 81 0.31 0.10 9 13 , 21 27 0.29 0.15 3 5 8 9 0.28 0.20 l 2 3 3 b__7 Commercial proprietary yogurt drink stabilizer 0.05 77 100 121 129 0.37 0.10 66 87 107 116 0.42 0.15 42 66 86 98 0.45 0.20 0 31 65 70 0.85 0.25 0 21 28 29 1.10 0.30 0 0 0 0 °--- aAverage of duplicate runs Sample was so stable that essentially no syneresis occurred c . No synere31s 70 Table 20. aSyneresis volume (ml) of yogurt drink samples after storage as a function of stabilizers at different concentrations. pH; 4.0, heat treatment; 88°C/2 min, homogenization pressure; 175 kg/cmz. Storage 0 Concen- (Days at 5 C) Remarks tration 3 5 8 10 Proteins(%) (mouthfeel) Propylene glycol alginate 0.05 70 85 91 96 0.38 0.10 48 61 70 79 0.35 0.15 30 42 60 73 0.32 0.20 35 46 61 72 0.31 Heavy Pectin 0.05 68 92 101 104 0.39 0.10 51 65 76 81 0.35 0.15 21 28 40 64 0.33 0.20 34 44 60 75 0.32 Heavy aAverage of duplicate runs. SYNERESISUIII) 71 250? o————«3 (L051 o————o Ilon 200~ A————¢. mtsx b-----‘ 0. 25 % 150 I 100 50 o 1 1 n 1 n . 2 4 6 8 10 STORAGE (Don ot 5°C) Figure 3. Syneresis volume of yogurt drink samples after storage as a function of different concentrations of carrageenan. pH; 4.0, heat treatment; 88°C/2 mig, homogenization pressure; 175 kg/cm . SYNERESISIMII 250' 200+ 15°F 100- Figure 4. 72 o————a» moss; o————<> o.u>% CL————U (115% AF-——43 0.ul% 2 4 O 8 1O STORAGE( Days at 5°C) Syneresis volume of yogurt drink samples after storage as a function of different concentrations of guar gum. pH; 4.0, heat treatment; 8809/2 min, homogenization pressure; 175 kg/cm . SYNERESIS ( ml) 73 250’ o————c «moss: o————o atom: 200», I—-I 0.1596 D———-fl doom. .b————dl 0.25% A_———4§ 0.30% 150? 100? 0 50+ 0 1 2 4 6 8 1O STORAGE(Doyo at 5°C) Figure 5. Syneresis volume of yogurt drink samples after storage as a function of different concentrations of commercial proprietary blended stabilizer. pH; 4.0, heat treatment; 88°C/2 mig, homo- genization pressure; 175 kg/cm . SYNERESIS (ml) 74 250' o————o ILO5‘5 o—-——<> 0 HI% A ----- 43 0.201. 150’ ‘OOP / C . -= o ",v 3 ‘ 50!- . ' ‘ ”'0’ ”’a’ I ’3' ’I I l l l l #1 ° 2 4 6 a » to STORAGE (Days at 5°C) Figure 6. Syneresis volume of yogurt drink samples after storage as a function of different concentrations of propylene glycol alginate. pH; 4.0, heat treat- ment; 88 C/2 min, homogenization pressure; 175 kg/emZ. SYNERESIS (ml) 75 250p .———. 0. 05 $ 0—0 0. 10 $ 200 - H o. 1 5 s A------A O. 20 i 150* 100 '- O O ’A . aérv"” ' 50 I- . ””'vooo o”' . I’IE’ I I I I I”” o ’ 1 l L I 4 6 8 10 Figure 7. STORAGEWoys at 5°C) Syneresis volume of yogurt drink samples after storage as a function of different concnetrations of pectin. pH; 4.0, heat treatment; 88°C/2 mi , homogenization pressure; 175 kg/cm . 76 Guar gum (Table 19, Figure 4) was found very effec- tive in stabilizing the system. Stability of the product increased with increase in its concentration until the product was essentially stabilized at the concentration of 0.20%. Notable stabilizing effect was observed when its concentration was raised from 0.05% to 0.10%. As can be seen from Table 19 and Figure 5, the commercial proprietary blended stabilizer exerted an ex- cellent stabilizing effect on the system. Stability of the product was enhanced increasingly with increase in its concentration until the product was finally stabil- ized at the concentration of 0.30%. Propylene glycol alginate (Table 20, Figure 6) and pectin (Table 20, Figure 7) showed almost identical stabil- izing pattern with that of carrageenan except that in this case the adverse effect on the product stability was ob- served at even lower concentration, 0.20%. At this con- centration, they also imparted apparent viscosity to the product. In summary, guar gum and a commercial proprietary blended stabilizer proved to be very effective in sta- bilizing yogurt drink, while carrageenan, propylene glycol alginate and pectin were less effective. The adverse effect on the stability and increased viscosity of the product caused by the higher conentrations (0.20% 77 or 0.25%) of those three stabilizers mentioned above in- dicate that these concentration were excessive for this particular product. Rasic and Kurmann (1978) stated that over-use of a stabilizer in dairy products not only has adverse effect on the stability of the product, but also imparts undesired viscosity to the product. The viscosity cuased may be due to the thickening effect of those sta- bilizers through their ionic interactions with mono-, di- or trivalent salts in the product. Carrageenan, alginate and pectin are known to have polyanionic nature with a negative surface charge and to form complexes with cationic salts. Guar gum is known to be virtually neutral and non-ionic. Schachat and Raymond (1960) stated that carrageenan forms complexes with potassium and ammonium ions, alginate with calcium and acids and pectin with calcium and sugar, all giving thickening (viscosity promoting) effect. Behavior of the commercial proprietary blended stabilizer can not be fully explained since detailed information on the conentration of each ingredient stabilizer was not available. Experience has shown that when two or more stabilizers are combined, the stabilizing characteristics of that combination are not predictable. So a trial and error method has been suggested to find a suitable formulation for the stability of a certain food product. Guar gum, being proven to be suitable stabilizer for yogurt drink, 78 mumcfimam Hoowam wcma>moum p 550 omoHsHHmo Hwnuoezxonumuo Epm umpw n mcsu oumowHQSp mo ommuo>¢m N>mom em.o oN oH HH N oH.o IIII IIII OH.o >>m0m om.o ON NH NH N IIII OH.o IIII 0H.o em.o NOH mm HN mm IIII IIII mH.o IIII N>mom mm.o Hm oN mH 0H IIII IIII 0H.o mH.o N>mom Hm.o HN NH HH N IIII IIII mo.o mH.o Nsmom Hm.o NH OH o e IIII IIII mo.o ON»o HHoomnosoeo HmoaHououm oH m m m sHuoom mom use we mxumfiom mI o muouflaenmum mo HUOm on usage Hwy cofluonucoocoo mmmuoum . EO\mx mNH Houpmmmum :oHumNNcomoEos .GHE «\UOmm HucwEuomHu upon .o.v mam .mGOHumuucoocoo ucmHoMMHp up mpcman HoNHHHnmum mo cofluogpm 8 mm ommuoum umumm moamsmm xcwup unpmow mo HHEV oEpHo> memouocmmm .Hm manna 79 was chosen to be tried in combination with CMC, alginate and pectin. The concentration of each stabilizer, syner- esis volume of each sample and protein content of the syneresed fluid after storage of 10 days are shown in Table 21. The comparison between the syneresis volumes appeared in Table 21 and those in Table 19 (guar gum only) indicates that the synergistic effect expected was not achieved by these combinations. Same concentration of guar gum caused more syneresis in all combinations tried when it was used with CMC than when it was used alone. And these combinations imparted apparent viscosity to the product. It is likely that the total concentration of these two stabilizers was excessive for this product due to the specific properties contributed by CMC which is also known to form complexes with cationic salts, thereby giving thickening effect. Stability of the product was improved slightly when guar gum was used in combination with alginate and pectin. However, these combinations also imparted viscosity to the product. This result may be due to the specific properties of alginate and pectin discussed previously. Data presented in Table 19-21 show that there is no correlation between the syneresis volume of the samples and its percentage protein content. The protein contents of all samples are in the range of 0.3410.06% except those of the samples for which commercial proprietary 80. blended stabilizer was used. It appears that much larger amounts of proteins were held in the suspension by the interaction with stabilizers even at the lower concentrations and that there were only smaller amount of proteins (which would be interacted by the higher concentrations of stabilizers) and smaller molecular ni- trogen compounds such as amino acids and urea in the syneresed fluid. Thus the slightly decreased percentage protein contents at the higher concentrations of stabili- zers may be attributable to the interactions of increased stabilizers with the small amounts of exudate proteins not interacted by stabilizers at the lower concentrations. Commercial blended stabilizer resulted in marked increase of exudate protein contents of the samples with increase of its concentration. It is most likely that all the proteins capable of being interacted by this stabilizer were interacted even at the lowest concentration (0.05%) of the stabilizer and the increased concentrations only enhanced its function of maintaining suspension and ab- sorbing free water, thus making the exudate more and more concentrated due to its decreasing volume. With the two samples which were finally stabilized by the use of guar gum (0.20%) and commercial stabilizer blend respectively, preference (acceptability) test was performed using a nine point hedonic scaling method. It was evident that there was no significant difference in 81 Table 22. Scores of the two samples finally stabilized by the use of stabilizers at the designated processing parameters . Stabilizers Commercial blended Judges Guar gum (0.20%) stabilizer (0.30%) Total 1 8 8 l6 2 9 9 18 3 8 7 15 4 8 j 8 16 5 9 8 l7 6 9 8 17 7 8 8 16 8 7 9 16 9 7 8 15 10 9 8 17 ll 8 7 15 12 8 6 14 13 7 7 14 14 9 8 17 15 7 8 15 16 8 8 16 17 9 8 17 18 8 9 17 19 9 6 15 20 4 8 12 21 8 8 16 22 4 7 ll 23 8 8 16 24 7 8 15 25 8 8 16 26 6 8 14 Total 200 203 403 apH 4.0 Heat treatment 88°C/2 min 2 Homogenization pressure 175 kg/cm 82 -preference for these two samples since the two total scores given by panelists were almost identical (Table 22). Therefor an analysis of variance was not conducted on these data. The mean values (total score/number of judges) calculated were 7.7 and 7.8 respectively. These values are close to "like very much (8)" on the hedonic scale. Both of the stabilized samples showed excellent acceptability. SUMMARY AND CONCLUSIONS The objectives of this research were to establish. optimum processing parameters and the concentration of suitable stabilizer(s) for the stability and sensory properties of yogurt drink. Yogurt drink was composed of 25% plain low fat yogurt, 10% frozen concentrated orange juice (4+1), 9% sugar and 56% water. Sedimenta- tion of syneresis in the samples on storage was observed and protein content was determined on the sample super- natant fluid. Subjective assessment of sensory proper- ties was conducted, being given the first priority for choosing optimum processing parameters. Subject to the conditions of this study, the following conclusions were derived: 1. Of the range of pH evaluated, pH 4.0 was the best in regard to flavor and stability of the product. Lower pH (3.2 and 3.6) resulted in excessive sourness. The sample with pH of 4.2 was acceptable, but a little too sweet. The sample with pH of 4.0 was more stable (less sedimentation and more proteins in the supernatant fluid) than the yogurt drink with pH of 4.2. 2. Heat treatment (65°C/30 min, 75°C/10 min, 88°C/2 min and 115°C/4 sec) had negligible effect on the sensory properties of the product. High temperature pasteuriza- tion improved stability of the product, which was con- siderably increased with increasing temperature of heat 83 84 treatment although there was little difference between the heat treatments of 65°C/30 min and 75°C/10 min. 3. Homogenization pressure (70 kg/cmz, 105 kg/cm2 and 175 kg/cmz) had little effect on the sensory proper- ties of the product. Low pressure homogenization favored stabilization of the product. Product stability was significantly decreased with increasing homogenization pressure. 4. Among the stabilizers used, carrageenan, alginate and pectin were ineffective in stabilizing the product under the conditions used in this project. When these stabilizers were used, the stability of the system dimished markedly with time and high concentrations (0.20% or 0.25%) imparted too much viscosity to the product. Guar gum and a commercial proprietary blended sta-- bilizer were very effective in stabilizing the system. Stability of the product increased with increasing con- centration of these stabilizers until the product was finally stabilized at the concentrations of 0.20% and 0.30%‘respective1y. The use of guar gum in combination with alginate, pectin and CMC was not effective in stabilizing this system. The synergistic effect of the stabilizers was not observed in any combinations studied. 85 5. Both samples which were finally stabilized using guar gum (0.20%) and a commercial blended stabilizer (0.30%) respectively showed excellent acceptability in taste panel evaluation, the mean values of both samples being close to 8 (like very much) on the nine point hedonic scale. APPENDIX 86 APPENDIX Statistical Charts Table 23. Rank totals required for significance at the 5% level (P< .05) No. of Number of tregtments _ Reps. 2 3 4 5 6 7 8 2 ................................... --------------- 3-8 3-11 3-13 4-14 3 --------------- 4-14 4-17 4-20 4-23 ----- 4-8 4-11 5-13 8-15 8-18 7-20 4 ----- 5-11 5-15 6-18 6-22 7-25 7-29 ----- 5-11 6-14 7-17 8-20 9-29 10-26 5 ----- 6-14 7-18 8-22 9-26 9-31 10-35 6-9 7-13 8-17 10-20 11-24 13-27 14-31 6 7-11 8-16 9-21 10-26 ll-3l 12-36 13-41 7-11 9-15 11-19 12-24 14-28 16-32 18-36 7 8-13 10-18 11-24 12-30 14-35 15-41 17-46 8-13 10-18 13-22 15-27 17-32 19-37 22-41 8 9-15 11-21 13-27 15-33 17-39 18-46 20-52 10-14 12-20 15-25 17-31 20-36 23-41 25-47 9 ll-18 13-23 15-30 17-37 19-44 22-50 24-57 ll-16 14-22 17-28 20-34 23-40 28-46 29-52 10 12-18 15-25 l7-33 20-40 22-48 25-55 27-63 12-18 16-24 19-31 23-37 26-44 30-50 33-57 Table 23 (cont'd.) 87 No. of Number of treatments Reps. 2 3 4 5 6 7 8 11 13-20 16-28 19-36 22-44 25-52 28-60 31-68 14-18 18-26 21-34 25-41 29-48 33-55 37-82 12 15-21 18-30 21-39 25-47 28-56 31-65 34-74 15-21 18-29 24-36 28-44 32-52 37—59 41-67 13 16-23 20-32 24-41 27-51 31-60 35-69 38-79 17-22 21-31 26-39 31-47 35-56 40-64 45-72 14 17-25 22-34 26-44 30-54 34-64 38-74 42-84 18-24 23-33 28-42 33-51 38-60 44-68 49-77 15 19-26 23-37 38-47 32-58 37-68 41-79 46-89 19-26 25-35 30-45 36-54 42-83 47-73 53-82 16 20-28 25-39 30-50 35-81 40-72 45-83 49-95 29-27 27-37 33-47 39-57 45-67 51-77 57-87 17 22-29 27-41 32-53 38-64 43-76 48—88 53-100 22-29 28-40 35-50 41-61 48-71 54-82 61-92 18 23-31 29-43 34-56 40-88 46-80 51-93 57-105 24-30 30-42 37-53 44-64 51-75 58-86 65-97 88 Table 24. Variance ratio-5 percent points for dis- tribution of F a ._ nl bn 1 2 3 4 5 6 8 2 1 161.4 199.5 215.7 224.6 230.2 234.0 238.9 2 18.51 19.00 18.25 19.25 19.30 19.33 19.37 3 10.13 9.55 9.28 9.12 9.01 8.94 8.84 4 7.71 6.94 6.59 6.39 6.26 6.16 6.04 5 6.61 5.79 5.41 5.19 5.05 9.95 4.82 6 5.99 5.14 4.76 4.53 4.39 4.28 4.15 7 5.59 4.74 4.35 4.12 3.97 3.87 3.73 8 5.32 4.46 4.07 3.84 3.69 3.58 3.44 9 5.12 4.26 3.86 3.83 3.48 3.37 3.23 10 4.96 4.10 3.71 3.48 3.33 3.22 3.07 11 4.84 3.98 3.59 3.36 3.20 3.09 2.95 12 4.75 3.88 3.49 3.28 3.11 3.00 2.85 13 4.67 3.80 3.41 3.18 3.02 2.92 2.77 14 4.60 3.74 3.34 3.11 2.96 2.85 2.70 15 4.54 3.68 3.29 3.06 2.90 2.79 2.64 16 4.49 3.63 3.24 3.01 2.85 2.74 2.59 17 4.45 3.59 3.20 2.96 2.81 2.70 2.55 18 4.41 3.55 3.16 2.93 2.77 2.68 2.51 19 4.38 3.52 3.13 2.90 2.74 2.63 2.48 20 4.35 3.49 3.10 2.87 2.71 2.60 2.45 21 4.32 3.47 3.07 2.84 2.68 2.57 2.42 22 4.30 3.44 3.05 2.82 2.66 2.55 2.40 23 4.28 3.42 3.03 2.80 2.64 2.53 2.38 24 4.26 3.40 3.01 2.78 2.62 2.51 2.36 25 4.24 3.38 2.99 2.76 2.60 2.49 2.34 26 4.22 3.37 2.98 2.74 2.59 2.47 2.32 27 4.21 3.35 2.96 2.73 2.57 2.46 2.30 28 4.20 3.34 2.95 2.71 2.56 2.44 2.29 29 4.18 3.33 2.93 2.70 2.54 2.43 2.28 30 4.17 3.32 2.92 2.69 2.53 2.42 2.27 89 Table 24 (cont'd.) a b n 1 2 3 4 5 6 8 n 2 40 4.08 3.23 2.84 2.61 2.45 2.34 2.18 60 4.00 3.15 2.76 2.52 2.37 2.25 2.10 120 3.92 3.07 2.68 2.45 2.29 2.17 2.02 m 3.84 2.99 2.60 2.37 2.21 2.09 1.94 aDegrees of freedom for numerator bDegrees of freedom for denominator LIST OF REFERENCES LIST OF REFERENCES Amerine, M.A., R.M. 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