機能性民族風味發酵乳之開發(2/3)

行政院國家科學委員會專題研究計畫成果報告

* * * * * * * * * * * * * * * * * * * * * * * * *

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機能性民族風味發酵乳之開發

*

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Development of national flavor fermented milk with

*

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physiological functionality

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* * * * * * * * * * * * * * * * * * * * * * * * *

計畫類別：特約研究員專案計畫

計畫編號：NSC 89-2313-B002-142

執行期間：88 年 8 月 1 日至 91 年 7 月 31 日

計畫主持人：林慶文

處理方式：一年後可對外提供參考

執行單位：台灣大學畜產系

中華民國 90 年 4 月 30 日

Microencapsulation of probiotics and chiu-niang extract in

1

Kou Woan Lao (an oriental style dairy product) by

2 spray-drying 3 4 5 6

Laboratory of Chemistry & Technology of Animal Products, Department

7

of animal science, National Taiwan University, Taipei, Taiwan, ROC

8 9 10 11 12

Key words: probiotics, lao -chao, Kou Woan Lao, microencapsulation

13 spray- drying, 14 15 16 17 18 19 20

*Corresponding author: Prof. Chin- Wen Lin

21

No. 50, Lane 155, Keelung Rd. Sec. 3,

22

Taipei, Taiwan, Republic of China

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Phone: 886-2-27336312

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Fax: 886-2-27324070

ABSTRACT

26

This objective was to combine the physiological functionality of

27

probiotics and the unique flavor and milk-clotting activity of culture

28

filtrate from lao-chao for the development of a new dairy product so

29

different from the commercial yogurt. Two probiotic strains,

30

Lactobacillus acidophilus and Bifidobacterium longum were applied to

31

skim milk before acid-curd formation, and then culture filtrates were

32

added for the formation of milk curd. The higher percentage of SNF, the

33

stronger curd firmness (P<0.05). The percentage of syneresis decreases

34

significantly when the level of SNF rising (P<0.05). As the SNF

35

increasing from 10% to 18%, the percentage of syneresis decreases from

36

12.9% to 4.3%. The pH value and probiotic counts of 1% L. acidophilus

37

group did not significantly decrease during 14 days storage at 4 ℃. The

38

counts of probiotics decrease about 1 log CFU/ml after spray-drying

39

process at 50℃ outlet temperature. The final total probiotic counts are

40

about 6.58 log CFU/ml. Regarding resuspension methods, it is found that

41

there are no significant difference of using stomacher or vortex to release

42

probiotics (P>0.05). The counts of probiotics significantly decrease when

43

the outlet temperature getting higher. Therefore, when the spray-drying

44

outlet temperature cools down, the survival percentage of probiotics

increases. In order to increase the probiotic counts of Kou Woan Lao, it is

46

suggested to choose the lower temperature of microencapsulation. In the

47

same time, the product quality and storage period could be effected due to

48

the spray-drying process. It will be a challengeable task to adjust different

49

production procedure in order to meet market needs.

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

INTRODUCTION

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The use of Lactobacillus acidophilus and/or Bifidobacterium spp. in

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fermented or culture-containing dairy products became popular by the

67

end of the 1970s as a result of the increase in knowledge encompassing

68

the properties of bifidobacteria. The nutritional benefits of probiotics

69

have been mostly studied in milk-based products fermented with

70

lactobacilli and bifidobacteria. They are characterized by a lower level of

71

residual lactose and higher levels of free amino acids and certain vitamins

72

than non-fermented milks. Furthermore, they preferentially contain

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L(＋)-lactic acid (that is more easily metabolized by human beings than

74

D(－)-lactic acid) produced by bifidobacteria in addition to acetic acid.

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Moreover, the L(＋)-lactic acid absorbed in the intestine is used as energy

76

source, with an energy yield of 15 kJ/g, that compares well with 16 kJ/g

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for lactose(Gurr, 1987). During the past years, numerous healthy and

78

nutritional benefits have been claimed including maintenance of normal

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intestinal microflora, alleviation of lactose intolerance, reduction of

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serum cholesterol levels, potential antitumor activity, and some

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therapeutic effects on intestinal disturbances and intestinal infections. The

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intact intestinal epithelium, together with the normal intestinal microflora

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represents a barrier to movement of pathogenic bacteria, antigens and

other noxious substances from the gut lumen to the blood. In healthy

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subjects this barrier is stable, thus protecting the host and assuring normal

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intestinal function. When either the normal microflora or the epithelial

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cells are disturbed, as triggered by dietary antigens, pathogens, chemicals

88

or radiation, defects in the barrier mechanisms become apparent; altered

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permeability facilitates blood invasion by pathogens, foreign antigens and

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other harmful substances (Gomes and Malcata, 1999). Experiments

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performed in animal models showed that a few strains of L. acidophilus

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and Bifidobacterium spp. are able to decrease the levels of enzymes

93

responsible for activation of some procarcinogens, and consequently

94

decrease the risk of tumor development. (Mital and Garg, 1992). A

95

number of studies indicate that administration of bifidobacteria or

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lactobacilli alone or with fermentable carbohydrate (defined as a prebiotic)

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can alter colonic microflora populations and decrease the development of

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early preneoplastic lesions and tumors. Many of the antitumor activities

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attributed to lactic cultures have been suggested to involve an enhanced

100

function of the immune response (Hirayama and Rafter, 2000). The

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anticarcinogenic, antitumorigenic, and antimutagenic activities of these

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probiotic bacteria have been proposed to occur via some modification of

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mutagens in vitro (Vorobeva et al., 1995), reduction in

carcinogen-generating fecal enzymes in vivo (Kulkarni and Reddy, 1994),

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and suppression of precancerous lesion or tumor formation (Reddy and

106

Rivenson, 1993).

107

A new oriental-style dairy product coagulated with culture filtrates

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from lao-chao (chiu-niang), a fermented rice product well known in

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China, has been developed to meet consumer preferences for a low-acid

110

or non-sour tasting yogurt-like product (Kuo et al., 1996 ; Lin and Chen,

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1996). Culture filtrates from lao-chao, which is produced through

112

fermentation by inoculating steamed glutinous rice with commercial

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starter (chiu-yao) or fungal cultures, have been used as both milk-clotting

114

agents and flavoring agents. The resultant yogurt-like products with soft

115

curd appearance are characterized by a sweet, fruity and slightly alcoholic

116

flavor. Therefore, this objective was to combine the physiological

117

functionality of probiotics and the unique flavor and milk-clotting activity

118

of culture filtrate from lao-chao for the development of a new dairy

119

product so different from the commercial yogurt.

120

Microencapsulation is a technique in which a membrane encloses

121

small particles of solid, liquid, or gas, with the objective of offering

122

protection to the sensitive core material from adverse environmental

123

conditions such as undesirable effects of light, moisture, and oxygen, thus

contributing to an increase in the shelf life of the product and promoting a

125

controlled liberation of the encapsulate (Shahidi and Han, 1993). Many

126

methods for preparing microcapsules have been developed and improved

127

significantly. They can briefly classify as mechanical and chemical

128

processes (Thies, 1996). Spray-drying is the microencapsulation

129

technique most widely used in the chemical, pharmaceutical, and food

130

industries (Gibbs, 1999). Spray-drying emulsions is a particularly

131

effective means of microencapsulating chemically reactive oils, volatile

132

oils, and flavor compounds. Spray-dried powder particles contain the

133

encapsulated material as minute droplets embedded within its wall

134

(McNamee et al., 1998).

135

Gum arabic (gum Acacia) is a hydrocolloid produced by the natural

136

excudation of arabic trees and is an effective encapsulation agent due to

137

its high water solubility, the low viscosity of concentrated solutions

138

relative to other hydrocolloid gums, and its ability to act as an

139

oil-in-water emulsifier. Gum arabic is composed of a highly branched

140

arrangement of the simple sugars galactose, arabinose, rhamnose, and

141

glucuronic acids (Street and Anderson, 1983) and also contains a protein

142

component (~2%w/w) covalently bound within its molecular arrangement

143

(Anderson et al., 1985). The protein fraction plays a crucial role in

144

determining the functional properties of gum arabic (Randall et al., 1988).

Maltodextrins and chemically modified starches have been investigated

146

as replacers for gum arabic in spray-dried emulsions (Amandaraman and

147

Reineccius, 1987). To wholly or partially replace gum arabic as an

148 encapsulating agent. 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165

MATERIALS & METHODS

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Preparation of inocula

167

The two strains of fungal cultures, Rhizopus javanicus (CCRC 30288)

168

and Saccharomyces cerevisiae (CCRC 21685), from the Culture

169

Collection and Research Center, Taiwan, R. O. C., were used to inoculate

170

steamed glutinous rice. Before use, R. javanicus and S. cerevisiae were

171

transferred to slants of YM agar and those of potato dextrose agar,

172

respectively, and incubated at 30±1 ℃ for 6-8 days. In addition, the

173

probiotics strains, Lactobacillus acidophilus (CCRC 14079) and

174

Bifidobacterium longum (CCRC 14605) were transferred to Lactobacilli

175

MRS (deMan, Rogosa and Sharpe) and MGL (modified Garche’s

176

lithium-chloride) media. Fungal spore suspensions and yeast cell

177

suspensions for inoculation were prepared by adding sterilized distilled

178

water containing 0.1 gL-1 Tween 80 to slants and shaking the cultures

179

vigorously for 1 min.

180

Preparation of fermented rice (lao-chao) and culture filtrates

181

Glutinous rice was purchased from a local market (Taipei, Taiwan,

182

R.O.C.). One hundred grams of glutinous rice, which had been washed

183

with distilled water and drained, was soaked in 75 ml distilled water at

184

room temperature (20-25℃) for 12 hr, sterilized at 121℃ for 15 min, and

185

then cooled to 35℃. The steamed glutinous rice was inoculated with 5 ml

186

suspensions containing 107 cells of S. cerevisiae and 107 spores of R.

javanicus and mixed completely, followed by static incubation at 30±1

188

℃ for the designated period. After fermentation, the culture filtrates were

189

obtained by filtering the fermented rice through 4 layers of cheesecloth.

190

The sediment in the culture filtrate was eliminated by centrifuging at

191

1,480×g for 30 min (Kubota, KR-20000T, Japan).The culture filtrates

192

were stored at 4 ℃ for further analysis.

193

Preparation of Kou Woan Loa and determination of viable probiotics

194

Pasteurized skimmilk contains various ratios of SNF was inoculated

195

with probiotics and then incubated at 37 ℃. The viable probiotics were

196

determined during the periods of incubation and storage (4℃ ) by

197

according to the method of Teraguchi et al.(1982).

198

Determination of the firmness of product

199

The firmness, as measured by the breaking force of the Kou Woan

200

Loa product, was determined by using a rheometer (Fudoh

201

NRM-2010J-CW, Tokyo 141, Japan) with a rheoplotter (Rikadenki Kigyo,

202

FR 801, Tokyo 113, Japan). Adaptor No.4 (20 mm diam.) of the

203

rheometer was used and table speed was 50 mm min-1.

204

Viscosity of Kou Woan Loa

205

The apparent viscosity was determined by using a Viscometer

206

(Brookfield, LVDV-II＋Viscometer, U. S. A.) with a No. 3 disc spindle at

207

a speed of 0.3rpm.

Encapsulation procedure

209

Preparation of probiotics and culture filtrates emulsion

210

All glassware, and solution used in the protocols were sterilized at

211

121 ℃ for 15 min. Gum arabic obtained from Hayashi Pure Chemical

212

Industries, Ltd. (Osaka, Japan). Maltodextrins and chemically modified

213

starches obtained from Gemfont Co. (Taipei, Taiwan, R.O.C.). Emulsion

214

with different gum arabic ratios were prepared and agitated with magnetic

215

stirrer bars for 30 min.

216

Spray-Drying the Emulsion

217

The emulsions were spray-dried using a laboratory mini spray drier

218

(model B-190, Buchi Co., Flawil, Switzerland) by control inlet air

219

temperature, compressed air for spray-flow, compressed air consumption

220

and feed rate to obtain outlet temperatures ranging from 50 to 60 ℃. The

221

residence time of the product was very low. The dried powders were

222

collected and analysis immediately or subsequently stored in sealed glass

223

bottles at 4 ℃.

224

Determination of probiotic viability in spray-dried powders

225

We assessed the viability of the probiotics in the inoculated

226

emulsions preparations before spray drying and in the resulting powders

227

by examining MRS and MGL pour plates after 72 hrs of incubation at

228

37°C (aerobically or anaerobically). To 1g of powder, 9ml of diluents

were added (1:10 dilution); the powders were allowed to rehydrate by

230

stomacher (Stomacher 400 Lab Blender, Sewad Medical, London, UK) at

231

normal speed (230 rpm 65%) for 1 min or by mixing with a vortex (Maxi

232

Mix II, Barnstead Thermolyne, Iowa, U.S.A.) for 1 min to release

233

microencapsulated probiotics and then diluted further with diluent, and

234

appropriate dilutions were pour plated.

235

Statistical analysis

236

Data were analyzed using the general linear model procedure of the

237

SAS software package (SAS/STAT, 1987), and Duncan’s multiple range

238

test (Montgomery, 1991) were used to detect differences between

239

treatment means, statistical significance was tested at the 5% level. All

240

experiments were replicated three times.

241 242 243 244 245 246 247 248 249

RESULTS & DISCUSSIONS

250

Two probiotic strains, Lactobacillus acidophilus (CCRC 14079) and

251

Bifidobacterium longum (CCRC 14605) were applied to skim milk before

252

acid-curd formation. Different culture ratios on total probiotic counts

253

during incubation period examined are shown in Fig. 1. In the beginning

254

of incubation period, the counts of 1% L. acidophilus was lower than the

255

other three treatments significantly (P < 0.05). When the single starter

256

treatment compares with the mix of 1% L. acidophilus and 1% B. longum

257

treatments, the counts of L. acidophilus was lower than the counts of B.

258

longum significantly (P < 0.05). Synergistic growth-promoting effects

259

between L. acidophilus and B. longum are occur. L. acidophilus is

260

beneficial to B. longum as the former organism acts as an oxygen

261

scavenger.

262

According preliminary screening tests for suitable fungal and yeast

263

cultures, Rhizopus javanicus (CCRC 30288) and Saccharomyces

264

cerevisiae (CCRC 21685) were selected as starters of lao-chao and the

265

resultant culture filtrates were used as the milk-clotting agents and

266

flavoring agents. Effects of various ratios of culture filtrate on pH value

267

and titratable acidity in Kou Woan Lao during incubation period are

268

shown in Fig. 2. As the incubation period increasing, the pH value

269

decreases and titratable acidity rising gradually since the number of

probiotics increase. However, when the percentage of culture filtrate

271

increases, the growth of probiotics is inhibited. As long as the percentage

272

of culture filtrate getting higher, the increasing level of the titratable

273

acidity decreases. If the percentage of culture filtrate was controlled in

274

40%, the titratable acidity was keeping in the same level. The acidity

275

increases significantly during the 30th to 36th incubation hours.

276

During the 18th –36th incubation hour, there are no significant

277

differences among the four SNF treatments on probiotic counts of Kou

278

Woan Lao (Fig. 3).

279

Fig. 4 shows the higher percentage of SNF, the stronger curd

280

firmness (P<0.05). When the SNF of skim milk increasing from 10% to

281

16%, the curd firmness grows from 18.9g to 44.6g. In the mean time, the

282

viscosity significantly increases from 12200cps to 29800cps (P<0.05). In

283

Fig. 5, the percentage of syneresis decreases significantly when the level

284

of SNF rising (P<0.05). As the SNF increasing from 10% to 18%, the

285

percentage of syneresis decreases from 12.9% to 4.3%.

286

These results are similar to the finding of Parnell-clunies et al.

287

(1988). According to Parnell-clunies et al. (1988), the curd firmness and

288

viscosity depend on if the structure of casein micelle is complete or not.

289

They also indicate the curd firmness has positive relationship with the

290

viscosity.

In order to produce better quality of Kou Woan Lao, the increase of

292

SNF will help to improve the curd firmness and viscosity. It is also

293

helpful to decrease the percentage of syneresis. However, in order to meet

294

consumer flavor expectation and lower the production cost, it is

295

suggested to keep SNF between 12 and 14%.

296

After adding culture filtrate, the total probiotics counts in other three

297

treatment groups decrease significantly (P<0.05), except the 1% L.

298

acidophilus group (Fig. 6). The counts of 1% B. longum group decrease

299

from 6.3 log CFU/ml to 6.0 log CFU/ml. The 1% B. longum group has

300

dramatically decreased when compared with other groups. The pH value

301

and probiotic counts of 1% L. acidophilus group did not significantly

302

decrease during 14 days storage at 4 ℃.

303

In Table 1, the counts of probiotics decrease about 1 log CFU/ml

304

after spray-drying process at 50℃ outlet temperature. The final total

305

probiotic counts are about 6.58 log CFU/ml. Regarding resuspension

306

methods, it is found that there are no significant difference of using

307

stomacher or vortex to release probiotics (P>0.05). It is attribute to the

308

high solubility of gum arabic (about 50 %).

309

The viability of probiotics differs in various spray-drying outlet

310

temperature showed as Fig. 7. The counts of probiotics significantly

311

decrease when the outlet temperature getting higher. Therefore, when the

spray-drying outlet temperature cools down, the survival percentage of

313

probiotics increases. However, it may cause the poor condition of

314

spray-drying process, which will decrease the efficiency of

315

microencapsulation and the stability of storge.

316

There are four separate phases of spray-drying methods : 1.

317

Nebulization of the solution in the form of an aerosol. 2. Contact of the

318

nebulized solution with the warm air. 3. Drying of the aerosol. 4.

319

Separation of the dried product and the air charged with the solvent.

320

During these process, the probiotics are easily vanished. In order to

321

increase the probiotic counts of Kou Woan Lao, it is suggested to choose

322

the lower temperature of microencapsulation. In the same time, the

323

product quality and storage period could be effected due to the

324

spray-drying process. It will be a challengeable task to adjust different

325

production procedure in order to meet market needs.

326 327 328 329 330 331 332 333

ACKNOWLEDGMENTS

334

This research was conducted using funds provided by the R.O.C.

335

Administrative Yuan’s National Science Committee, Plan NSC–

336

89-2313-B002-142 Subsidy. We express our gratitude.

337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354

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355

Amandaraman, S. and G. A. Reineccius. 1987. Analysis of encapsulated

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orange peel oil. Perfum. Flavor. 12:33-39.

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Anderson, D. M. W., J. F. Howlett, and C. G. A. McNab. 1985. The

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amino acid composition of the proteinaceous component of gum

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arabic (Acacia senegal (L.)) Willd. Food Addit. Contam. 2:159-164.

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Gibbs, B. F., S. Kermasha, I. Alli, and N. Mulligan. 1999. Encapsulation

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in the food industry : a review. Int. J. Food Sci. Nutr. 50:213-224.

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Gomes A. M. P. and F. X. Malcata. 1999. Bifidobacterium spp. and

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Lactobacillus acidophilus : biological, biochemical, technological and

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Technol. 10:139-157.

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Gurr, M. I. 1987. Nutritional Aspects of Fermented Milk Products. FEMS

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Microbiol. Rev. 46:337-342.

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Hirayama, K. and J. Raffter. 2000. The role of probiotic bacteria in cancer

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prevention. Microbes and Infection 2:681-686.

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Kulkarni, N. and B. S. Reddy. 1994. Inhibitory effect of Bifidobacterium

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a yoghurt-like product. Food Chem. 55:129-131.

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Lin, C. W. and H. L. Chen. 1996. Screening and characterization of

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McNamee, B. F., E. D. O’Riordan, and M. O’Sullivan. 1998.

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Emulsification and microencapsulation properties of gum arabic. J.

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Agri. Food Chem. 46:4551-4555.

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Montgomery, D. C. 1991. Experimented with a single factor : the analysis

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Montgomery(Ed.), p.75-77. John Wiley & Sons, New York.

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Parnell-clunies, E., Y. Kakuda, and J. M. Deman. 1988. Gelation profiles

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yogurt as affected by heat of milk. J. Dairy Sci. 71:582-588.

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Randall, R. C., G. O. Phillips., and P. A. Williams. 1988. The role of the

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Shahidi, F. and X. Q. Han. 1993. Encapsulation of the food ingredients.

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411

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64:187-192.

Fig. 1. Effects of different culture ratios on total probiotic counts in skim milk during incubation period (n = 9 ; error bars indicate 95% confidence intervals).

LA : L. acidophilus. BL : B. longum.

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

0

6

12

18

24

30

36

Incubation time (hr)

Probiotic counts (log CFU/ml)

1%LA

0.5%LA+0.5%BL

0.3%LA+0.7%BL

1%BL

Fig. 2. Effects of culture filtrate on pH value and titratable acidity in Kou Woan Lao during incubation period (n = 9 ; error bars indicate 95% confidence intervals).

*CF : culture filtrate.

Fig. 3. Effects of solids-not-fat on probiotic counts in Kou Woan Lao during incubation period (n = 9 ; error bars indicate 95% confidence intervals).

*A : L. acidophilus .

B : B. longum .

0 10000 20000 30000 10 12 14 16 SNF (%) Viscosity (cps) 0.0 10.0 20.0 30.0 40.0 50.0 60.0 Curd firmness (g) Viscosity Curd firmness

Fig. 4. Effect of SNF content on curd firmness and viscosity of

0

2

4

6

8

10

12

14

16

18

10

12

14

16

18

SNF (%)

Syneresis (%)

Fig. 5. Effect of SNF content on syneresis of curd (n = 5 ; error

5.6 5.8 6.0 6.2 6.4 6.6 6.8

Probiotic counts (log CFU/ml)

1%LA 0.5%LA+0.5%BL 0.3%LA+0.7%BL 1%BL

Fig. 6. Effects of different culture ratios on total probiotic counts and pH value in Kou Woan Lao during refrigeration period (n = 9 ; error bars indicate 95% confidence intervals). 5.6 5.8 6.0 6.2 6.4 6.6 6.8

Probiotic counts (log CFU/ml)

1%LA 0.5%LA+0.5%BL 0.3%LA+0.7%BL 1%BL

5.6 5.8 6.0 6.2 6.4 6.6 6.8 0 2 4 6 8 10 12 14

Refrigeration period (days)

Table 1. The viability of probiotics during the spray-drying process (outlet temperature : 50 ℃)

Viability

L. acidophilus B. longum Total probiotic counts

Log (CFU / ml)

Emulsions (before spray-drying) 7.46 a±0.05 6.99 a±0.06 7.59 a±0.04

stomacher 6.45 b±0.03 5.99 b±0.01 6.58 b±0.02

After spray-drying

vortex 6.46 b±0.02 5.98 b±0.03 6.58 b±0.02

Fig. 7. Effect of outlet temperature on probiotic counts during the spray drying (n = 4 ; error bars indicate 95% confidence intervals).

L. acidophilus 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 before spray-drying after spray-drying B . l o n g u m 4 . 5 0 5 . 0 0 5 . 5 0 6 . 0 0 6 . 5 0 7 . 0 0 7 . 5 0 8 . 0 0 8 . 5 0 5 0 5 5 6 0 outlet temperature (℃) before spray-drying after spray-drying