FERMENTED FOOD PRODUCTS: I. INFLUENCE OF SOME FOOD ADDITIVES ON THE GROWTH AND ACID DEVELOPMENT OF LACTIC ACID STARTER AND PROBIOTIC BACTERIA Amira M. El-Kholy1, A. A. Shatta2 and M. M. Osman1* 1
Dairy Dept. and 2Food Technology Dept., Fac. of Agriculture, Suez Canal University, Ismailia 41522, Egypt. *Corresponding author: E-mail:
[email protected]. SUMMARY The growth of seven strains of lactic acid starter bacteria (Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus plantarum, Lactobacillus helveticus and Lactobacillus fermentum) and nine strains of probiotic bacteria (Lactobacillus acidophillus La-5, Lactobacillus casei strain Immunitas, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium bifidum Bb-11, Bifidobacterium lactis Bb-12, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium aldolescentis) was studied in liquid media in relation to some substances used with fermented food products. The substances used were sugar (sucrose), sweeteners (acesulfame-k, aspartame), salts (sodium chloride, potassium chloride), flavouring agents (mango, apricot, guava, strawberry, orange, lemon, green apple), colouring agents (raspberry, annatto, carmosine, caramel) and flavouring-colouring agent (vanillin). Sucrose, acesulfame-k and colouring agents did not interfere with the growth and acid development of the strains at concentrations commonly used in the food industry, while the effects of aspartame, sodium chloride, potassium chloride, flavouring agents and vanillin were concentration and strain dependent. Therefore, the tolerance of lactic acid starter and probiotic bacteria to food additives should be taken into consideration. Keywords: fermented food, Starter, Probiotic, Food additives, Lactic acid bacteria.
INTRODUCTION Fermented dairy products have been consumed for a very long time as highly nutritive foods and to maintain good health. The overall popularity of these products has expanded over the last decade. The use of bioproducts food has increased significantly, presumably because of its perceived health benefits (Wood, 1992). Furthermore, almost 100 bioproducts containing Lactobacillus acidophilus and bifidobacteria are available on the markets world wide (Dave and Shah, 1996). Several health and nutritional benefits have been ascribed to bifidobacteria and lactic acid bacteria including maintenance of desirable intestinal flora (Ouwehand et al., 1999), synthesis of vitamin B complex and absorption of calcium (Deguchi et al., 1985), Proc. The 1st International Conf. "Food for Better Health", NRC, 18-20 October (2003), Cairo, Egypt
El-Kholy et al.
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amelioration of diarrhea or constipation (Shornikova et al., 1997; Hilton et al., 1997), antimicrobial production (Kang et al., 1989) and immunity activation (Madara, 1997). Probiotic bacteria also lower the levels of fecal bacterial enzymes, responsible for catalyzing the conversion of carcinogenic amines (Spanhaak et al., 1998), improve lactose digestion and improve protein metabolism (Hughes and Hoover, 1995) and reduce serum cholesterol levels (Tahri et al., 1995). Potential application of culture blends containing probiotic bacteria includes sour cream, buttermilk, yogurt, powdered milk, spreads, frozen desserts, fruit juices, mayonnaise, dry fermented sausages, fermented meats or fish, deep fried LAB-fermented carrot chips, Japanese miso-fermented rice or soya been, fermented mackerel minces and peanut butter (Khalil and Mansour, 1998; Aukrust et al., 1994; Gab-Alla and Gad, 2001; Yin et al., 2002; Pszczola, 2002 and Gordon, 2002). In order to exert positive health effect, it is generally assumed that the microorganisms must be viable, active and abundant (at least 10 7 cfug-1) in the product to the date of minimum durability (International Dairy Federation, 1997). Selection of probiotics is based on medical, scientific and technological criteria (Collins et al., 1998), must tolerate the manufacturing processes and maintain cell viability during storage (Charteris et al., 1998). Strain survival in the product depends on many factors such as pH, presence of preservatives (Charteris et al., 1998) and even the occurrence of potential microbial growth inhibitors (Collins et al., 1998). The effect of additives commonly used by food industry on the growth of lactic acid starter and probiotic has been subject of several studies (Samona and Robinson, 1993; Gomes et al., 1998; Rada and Dlabal, 1998; Vachon and Ustunol, 1998; Vinderola et al., 2002) but further information is still needed. The aim of this study was to evaluate the influence of some food additives on the growth and acid development of lactic acid bacteria and probiotics.
MATERIALS AND METHODS Bacterial strains Lactococcus (Lc.) lactis ssp. lactis, Lc. lactis ssp. cremoris and Streptococcus (S.) thermophilus, Lactobacillus (Lb.) delbrueckii ssp. bulgaricus, Lb. helveticus, Lb. acidophillus La-5, Bifidobacterium (B.) bifidum Bb-11 and B. lactis Bb-12 were from Chr. Hansen’s Lab., Denmark. Other bacteria used in this study were Lb. plantarum (DSM 20205), Lb. fermentum (3025162 1M, FDRCK, Germany), Lb. casei strain Immunitas (isolated from biofermented milk), Lb. casei (LS/B/ 32 Wū), Lb. paracasei (DSM 5622), B. infantis (ATCC 15637), B. longum (ATCC 15707) and B. aldolescentis (ATCC 15704). Additives Different food additives were examined at the concentrations used in food industry: sucrose (Merck, Darmstadt, Germany) at 5 and 25% w/v; sweeteners aspartame and acesulfame-k (Hoechst, Aktingesellschaft, Frankfurt, Germany) at 0.04 and 0.15% w/v;
Influence of food additives on lactic acid starters
29
sodium chloride and potassium chloride (Merck) at 2 and 6% w/v. Flavouring agents, apricot A 63105 0.1% at w/v and green apple R 9410G at 0.017% w/v were from Delta aromatic company, Egypt; mango 7441/A at 0.033% w/v, orange EM1104 and lemon at 0.0625% w/v for each from Futura Labs International, Egypt; guava 4342 YSE at 0.01% w/v from Union Perfumery Aromatic and Cosmetics, 6 October City, Egypt and strawberry BC 101 at 0.005% w/v from Kamena Company, Cairo, Egypt. Colouring agents, carmosine 610 at 0.033% w/v and raspberry red (neococosin 16255) at 0.034% w/v were from Kamena Company, Cairo, Egypt; annatto at 0.021% w/v from Futura Labs International, Egypt and caramel E 150 98001 at 0.0125% w/v from Delta aromatic company, Egypt. Flavouring-colouring agent, vanillin at 0.1, 0.5 and 1.5 % w/v was from the Zhonghua Chemicals, Zhejiang, China. Culture media MRS broth medium (Biolife) was used as a growth medium for lactobacilli and bifidobacteria, while M17 broth medium (Biolife) was used for lactococci and streptococci. Methodology and incubation conditions Solution of food additives were sterilized by filtration through sterile 0.45 µm cellulose nitrate filter (Sartorius, AG. 37070 Goettingen, Germany) food additives and then added at different concentrations (mentioned above) to the bacterial growth media. The media were inoculated with 1%, v/v of bacterial culture (initial count, 10 6 to 107 cfu mL-1). The inoculated media were incubated at 37°C, aerobiosis for lactococci, streptococci, lactobacilli and anaerobiosis for bifidobacteria (GasPak System-Oxoid, Basingstoke, Hampshire, England). The growth was measured by absorbance at 600 nm on a Spectronic 20D spectrophotometer (Milton Roy Company, USA) at intervals (0, 3, 6, 12, 24, 48 and 72 hrs). The growth were expressed as normal growth (A600nm > 70% of the control culture), weak growth (30% < A600nm < 70% of the control culture) and no growth (A600nm 70% of the control culture).
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Table (2): Growth and pH value of probiotic bacteria in liquid media (37°C for 3 days) in the presence of different colouring agents Bacteria
Lb. acidophillus La-5 Growth pH Lb. casei strain Immunitas Growth pH Lb. casei Growth pH Lb. paracasei Growth pH B. bifidum Bb-11 Growth pH B. lactis Bb-12 Growth pH B. infantis Growth pH B. longum Growth pH B. aldolescentis Growth pH
Controla
Raspberry red
Anatto
Carmosine
Caramel
+ 4.37
+ 4.08
+ 4.20
+ 3.91
+ 4.21
+ 3.58
+ 3.67
+ 3.60
+ 3.66
+ 3.65
+ 4.32
+ 4.30
+ 4.29
+ 4.25
+ 4.28
+ 5.00
+ 5.08
+ 5.13
+ 4.14
+ 4.13
+ 4.47
+ 4.65
+ 5.33
+ 4.43
+ 4.51
+ 4.61
+ 4.73
+ 4.65
+ 4.20
+ 4.26
+ 3.75
+ 3.83
+ 3.77
+ 3.83
+ 3.93
+ 3.67
+ 3.71
+ 3.65
+ 3.70
+ 3.73
+ 3.67
+ 3.75
+ 3.68
+ 3.72
+ 3.72
a: In MRS broth medium without additives. +: Normal growth (A600 nm > 70% of the control culture).
22
Influence of food additives on lactic acid starters
2.5
Absorbance at 600 nm
2.0
1.5
1.0 Control of S. thermophilus
S. thermophilus Control of Lb. plantarum Lb. plantarum Control of Lb. helveticus Lb. helveticus
0.5
0.0 0
24
48
72
Time (hours)
Fig. (1): Effect of aspartame (0.15%) in liquid media on the growth of lactic acid starter at 37oC
4.0 Control of Lb. casei
Lb. casei
Absorbance at 600 nm
3.5
Control of Lb. acidophilus La-5 Lb. acidophilus La-5 Control of B. bifidum Bb-11 B. bifidum Bb-11 Control of B. lactis Bb-12 B. lactis Bb-12
3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
24
48
72
Time (hours)
Fig. (2): Effect of aspartame (0.15%) in MRS broth medium on the growth of probiotic bacteria at 37oC
011
El-Kholy et al.
2.5
Absorbance at 600 nm
2.0
1.5 Control of B. bifidum Bb-11 B. bifidum Bb-11 Control of Lb. paracasei
Lb. paracasei
1.0
0.5
0.0 0
24
48
72
Time (hours)
Fig. (3): Effect of NaCl (2%) in MRS broth medium on the growth o of probiotic bacteria at 37 C
3.5 Control of Lb. paracasei
Lb. paracasei Control of Lb. acidophilus La-5 Lb. acidophilus La-5 Control of B. bifidum Bb-11 B. bifidum Bb-11
Absorbance at 600 nm
3.0
2.5
2.0
1.5
1.0
0.5
0.0 0
24
48
72
Time (hours)
Fig. (4): Effect of NaCl (6%) in MRS broth medium on the growth of probiotic bacteria at 37oC
010
Influence of food additives on lactic acid starters
2.5
Absorbance at 600 nm
2.0
1.5
1.0 Control of Lb. acidophilus La-5 Lb. acidophilus La-5 Control of B. bifidum Bb-11 B. bifidum Bb-11
0.5
0.0 0
24
48
72
Time (hours)
Fig. (5): Effect of KCl (6%) in MRS broth medium on the growth of probiotic bacteria at 37oC
2 . 5
2 . 0
C o n t r o l A p r i c o t G u a v a
1 . 5
Absorancet60nm
1 . 0
0 . 5
0 . 0 0
2 4
4 8
7 2
T i m e ( h o u r s )
F i g . ( 6 ) : E f f e c t o f f l a v o u r i n g a g e n t s i n M R S b r o t h m e d i u m o n t h e g r o w t h o f o B b 1 1 a t 3 7 C
B .b if id u m
019
El-Kholy et al.
2.5
Absorbance at 600 nm
2.0
1.5 Control of Lb. plantarum Lb. plantarum (0.1%) Lb. plantarum (0.5%) Lb. plantarum (1.5%) Control of Lb. helveticus Lb. helveticus (0.1%) Lb. helveticus (0.5%) Lb. helveticus (1.5%)
1.0
0.5
0.0 0
24
48
72
Time (hours)
Fig. (7): Effect of vanillin% in MRS broth medium on the growth of o lactic acid starter at 37 C
2.5
Absorbance at 600 nm
2.0
1.5
Control of Lb. acidophilus La-5 Lb. acidophilus La-5 (0.1%) Lb. acidophilus La-5 (0.5%) Lb. acidophilus La-5 (1.5%) Control of B. bifidum Bb-11 B. bifidum Bb-11 (0.1%) B. bifidum Bb-11 (0.5%) B. bifidum Bb-11 (1.5%)
1.0
0.5
0.0 0
24
48
72
Time (hours)
Fig. (8): Effect of vanillin% in MRS broth medium on the growth of Lb. acidophilus La-5 and B. bifidum Bb-11 at 37oC
019
Influence of food additives on lactic acid starters
2.5
Absorbance at 600 nm
2.0 Control of Lb. casei strain Immunitas Lb. casei strain Immunitas (0.1%) Lb. casei strain Immunitas (0.5%) Lb. casei strain Immnuitas (1.5%) Control of Lb. paracasei Lb. paracasei (0.1%) Lb. paracasei (0.5%) Lb. paracasei (1.5%)
1.5
1.0
0.5
0.0 0
24
48
72
Time (hours)
Fig. (9): Effect of vanillin% in MRS broth medium on the growth of Lb. casei strain Immunitas and Lb. paracasei at 37oC
019
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منتجات األغذية المتخمرة -1 :تأثير بعض اإلضافات الغذائية على نمو إننتواا الموامض لئاتئوات حامض الالكتيك إالئكتريا المي ية 1
أميرة دمحم الخ لى ،1عاتل أب بكر شطا 2إمجدى دمحم عثمان
قسووما األلئووان 1إالصووناعات الغذائيووة – 2كليووة الاعاعووة – جامعووة قنوواة السو ي جمه عية مصر العربية
– اإلسووماعيلية – 21522
درستتا عنقة تتن وتتدٍ عضتتلد وقتتل عنًتتلعد عنيتتا متتترة نلى نتتن عنًيمًتتاد اهتتا ًَتتل ست قن يتتٍ ورد تتر حتريل عنةتيدت( Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, )Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus fermentum عمسقن يٍ عن كيانر عنحدلنن Lactobacillus acidophillus La-5, Lactobacillus casei strain Immunitas, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium bifidum Bb-11, Bifidobacterium lactis Bb-12, Bifidobacterium infantis, ) Bifidobacterium longum, Bifidobacterium aldolescentisع د عخي ا عإلضرفر عنغ ع دن عآلمدن :عنسكاعز ،يحهدر عالسدلسهفرو-ك ععألست ررمرو) ،ميتة( تهلرنتد عنيتلدنلو عتهلرنتد عن لمرستدلو)، يكس ر عنطقى يرَجل ،يشًش ،ضلعفتن ،فاععنتن ،واملترل ،ندًتلٌ ،مفتر( مختتا) ،يهلَتر عنيتل عن تا ، عألَرمل ،عنكرريلسدٍ ،عنكاعيدم) عيهلٌ عيكسب طقى عنفرَدهدٍ). نتتى متتبعا وق تل عنًتتلعد عنًتتترفن عنستتكاعز ،عالسدلستتهفرو-ك ععنًهلَتتر ) وتترنياتداع عنًستتيمدين فتتا عنيتتُران اهتتا ًَتتل عاَيتتر عنحتتريل نه كيانتتر عنًمي تتاد .ميتتر ولدتتن عنًتتلعد عألختتا عألستت ررمرو ،تهلرنتتد عنيتتلدنلو عتهلرنتتد عن لمرستتدلو ،يكس ت ر عنطقتتى ععنفتترَدهدٍ) فلتتد مايًتتد مهعدااتتر اهتتا ماتدااتتر ععنس تةنن عنًمي اد. نت ن( نجتتب مٌ نهخت فتتا عالاي ترر فتتا يجترل يتتُران عألى نتن عنًيمًتتاد يتد يغرن تتن ات ِ عن كيانتتر ن ت ِ عإلضرفر عنغ ع دن عنيا د نكلٌ ن ر مهعدا اكسا اها ًَل عاَير عنحريل ن رد ر وكيانر حتريل عنةتيدت( ععن كيانر عنحدلنن.
