NON-DAIRY PROBIOTIC BEVERAGE : APPLICATION OF Lactobacillus plantarum EMI AND Lactobacillus pentosus EMI CULTURES AS INOCULUM IN FERMENTED LEGUMES AND SWEET CORN MILK - Unika Repository

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The 4 th International Conference of Indonesian Society Lactic Acid Bacteria (ISLAB). Yogyakarta, 25th – 26th January 2013

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NON-DAIRY PROBIOTIC BEVERAGE :

APPLICATION OF Lactobacillus plantarum EM1 AND Lactobacillus pentosus

EM1 CULTURES AS INOCULUM IN FERMENTED LEGUMES AND SWEET CORN MILK

Binardo Adi Seno1, Amelia Juwana1, Lindayani1, and Laksmi Hartayanie1

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Post Graduate Program of Food Technology Department - Soegijapranata Catholic University, Semarang

Email : [email protected]

ABSTRACT

Probiotic means living microorganisms when in adequate amounts confer a health benefit. Most of the probiotic foods are dairy products, so they cannot be consumed by humans who are allergic to milk proteins, have severe lactose intolerance or vegetarian way of diet. Consequently, “milk” from legumes and cereal has become a very interesting food because they have excessive nutritive value and health characteristics. This research used four fermented probiotic beverage, i.e. three based on legumes products (soybean, mung bean and kidney bean) and sweet corn milk. The products were fermented for 40 days with selected lactic acid bacteria Lactobacillus plantarum

strain EM1 and Lactobacillus pentosus strain EM 1, which has been isolated from “sayur asin” (Indonesian fermented vegetables). The results showed that the total amount of lactic acid bacteria (LAB) in all samples have met the standard to be probiotic beverage according to National Standard of Indonesia (2009) and also CODEX STAN (2003). LAB viability in the storage is fairly stable, which L. plantarum

EM1 has the higher viability (65.16%-97.52%) than L. pentosus EM1 (58.89-96.72%), but two types of strain are not significantly different with the L.bulgaricus as a control (70.75%-98.76%). Biochemical changes occurred until 40 days storage and cause the difference of the probiotic beverage characteristics. After storage for 40 days, fermented sweet corn with L. plantarum EM1 as inoculum contained the highest LAB 3.9 x 106 CFU/ml, the highest LAB viability 90.65%, pH 4.24, sugar content 7.53°brix, total acid 0.54% and protein content 2.75%. Based on the results, were concluded that L.

plantarum EM1 and L. pentosus EM1 can be utilized as inoculum in all probiotic

beverages.

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1. Introduction

The term “probiotic” means living microorganism, which in sufficient numbers exert health benefits on the host. Clinical evidence provided by maintaining or improving their intestinal microbial balance. These microorganisms, mainly Lactobacilli and/or Bifidobacteria have numerous health promoting effects and also strengthening of the immune system to prevent many diseases. Some health-related effects from probiotics were managing lactose intolerance, prevention of colon cancer, reduction of cholesterol and triacylglycerol plasma concentration, lowering blood pressure, reducing inflammation and allergic symptoms and also suppression of pathogenic microorganisms (Çakır 2003, Scherezenmeir and De Vrese 2001, Dunne, et al. 2001, Dugas, et al. 1999 in Yavuzdurmaz, H. 2007). Furthermore, World Health Organisation (WHO) deemed probiotics to be the next most important immune defense systems because of its antibiotic resistance ability which significantly increased in recent years (Bengmark 2000 in Wadher, K.J., Mahore, J.G & Umekar, M.J, 2010).

Most of the commercially probiotics are milk-based fermented foods, which mostly cannot be consumed by people who are allergic to milk proteins, have severe lactose intolerance or vegetarian way of diet. Potential substrate sources to produce non-dairy probiotic beverages are legumes and cereals, which now have been improved and processed as “milk”. In nutrition, legumes have highly nutritional compound, for example richness in protein, essential fatty acid especially unsaturated fatty acids, soluble and insoluble dietary fibers, vitamin and minerals. Moreover, legumes have functional compounds like isoflavones in soybeans as antioxidants (Božanić, 2006) and oleanolic acid in mung beans as antioxidants and immune systems (Somova, 2003 & Raphael and Kuttan, 2003). Sweet corn is also highly nutritional compounds especially high in dietary fiber, niacin & folate and also some minerals. Recently, corn milk processed from sweet corn by pasteurized or UHT treatment is popular because the attractive color, aroma and appearance, together with the sweetness of the corn milk, are the main sensory characteristics that are sought by its consumers (Supavititpatana, 2010).

This study aims to develop some probiotic products based on legumes and sweet corn milk using LAB which has been isolated from “sayur asin” (Indonesian fermented vegetables). The preliminary studies have successfully isolated LAB from “sayur asin” which produced from the fermentation of “sawi pahit” (Brassica juncea var. Czern) (Evelyne, 2011). In this study, the changes of LAB population, the cell viabillity during storage at 4 C and some major chemical characteristic in probiotic based legumes and sweet corn milk were assessed.

2. Material and Methods

2.1. Preparation of Legumes & Sweet Corn Milk

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3 with 2 % sugar. After that, added with skim milk 5% and gelatin 1%, boiled with mild heat and stirred 5 minutes. (Widowati & Misgiyarta, 2003).

2.3. Starter Culture Preparation

Bacteria which used to make the probiotic beverages are Lactobacillus

plantarum EM1, Lactobacillus pentosus EM1 (isolated from “sayur asin” from

preliminary study), and Lactobacillus bulgaricus as a control. All bacteria were suspended with aquadest with OD around 0.1-0.2 nm. Bacteria suspensions were put into legumes and sweet corn based milk in ratio 1:4 for each inoculum (40 ml legume / sweet corn based beverages added with 10 ml inoculum), and incubated at 37oC for 24 hours (Usmiati & Utami, 2008 with modification).

2.4. Preparation of Probiotic Drink from Legumes and Sweet Corn Milk

Legumes and sweet corn based milk which have been cooked poured into 15 erlenmeyer 300 ml and then added with inoculum for 3% (3 ml) and incubated for 24 hours under room temperature (37º C). After incubation, the probiotic milk were stored in refrigeration temperature (4º C) for about 40 days. These probiotic milk were tested after incubation and during storage every 10 days until day 40 storage. All of probiotic beverage will be tested the amount of Lactic Acid Bacteria contained by standard plate count, the viability of Lactic Acid Bacteria and biochemical changes for storage period (pH, sugar contents and protein contents)

2.5. Enumeration of Lactic Acid Bacteria

MRS agar was used for the enumeration of lactic acid bacteria. One milliliter of appropriate serial dilutions of each samples were pour-plated onto the media (surface platting method). After 48 h of incubation at 37⁰C, the colonies on the plates were counted based on Standard Plate Count (SPC) and the CFU/ml were calculated. (Hadioetomo, 1993 with modification)

2.6.Viability Test of Probiotic Bacteria

The viability test was done in MRS agar media using surface plating method with vary of dilution series. Viability of probiotics was counted based on logarithmic ratio on total bacteria per ml before and after storage and was presented in percent (%). The equation was:

Viability (%) = 100%

) /

(log

) /

(log

x storage before

ml cfu

storage after

ml cfu

(Rizqiati et al., 2008).

2.7. Chemical Analysis

Chemical analysis involved pH measurements and titratable acidity (Chandan et al., 2006 with modification and Wijaningsih, 2008), sugar contents by refractometer (Nielsen, 1998) and protein contents (AOAC, 1995).

3. Result and Discussion

3.1.Total Lactic Acid Bacteria in Legume and Sweet Corn Based Milk

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4 from day 1 until day 30 storage although the amount of bacteria decreased during storage. According to National Standard of Indonesia (Standar Nasional Indonesia or SNI) number 7552: 2009 and CODEX STAN 2003, the minimum amount of bacteria for fermented milk must reach 1x106 CFU/ml. Therefore, both legume and sweet corn based milk have met the standard to be probiotic beverage for all cultures (L.bulgaricus,

L.plantarum EM1, L.pentosus EM1). During storage, the total lactic acid bacteria are

decreased, may be due to the storage temperature is not fully inhibit the activity of the metabolism so that the availability of nutrients in the medium diminished and unable to support further growth of this bacteria. Lidya and Djenar, (2000) in Sumarsih et al., (2011), added the decrease caused by the growing accumulation of toxic metabolic results of bacteria cells.

Table 1.Total Lactic Acid Bacteria of Soy Bean Based Milk

Day Total Lactic Acid Bacteria (CFU/ml) in Soy Bean Milk

L. bulgaricus L. plantarum EM1 L. pentosus EM1

1 1.56 x 108 2.56 x 108 3.06 x 108

10 2.52 x 107 1.97 x 107 2.28 x 107

20 1.46 x 107 1.64 x 107 1.91 x 107

30 3.70 x 106 4.90 x 106 1.70 x 106

40 1.10 x 106 3.00 x 105 1.00 x 105

Source: Yani, 2012

Table 2.Total Lactic Acid Bacteria of Mung Bean Based Milk

Day Total Lactic Acid Bacteria (CFU/ml) in Mung Bean Milk

1 1.84 x 107 2.23 x 107 2.11 x 107

10 1.85 x 107 1.02 x 107 1.21 x 107

20 5.90 x 106 7.70 x 106 7.30 x 106

30 1.20 x 106 2.20 x 106 3.90 x 106

40 9.00 x 105 1.60 x 106 5.00 x 105

Source: Dewi, 2012

Table 3.Total Lactic Acid Bacteria of Kidney Bean Based Milk

1 2.40 x 107

2.75 x 107

2.40 x 107

10 1.29 x 107

1.58 x 107

5.89 x 106

20 8.71 x 106 9.77 x 106 4.79 x 106

30 4.90 x 106

6.46 x 106

3.31 x 106

40 3.24 x 106

3.02 x 106

1.00 x 105

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5 Table 4.Total Lactic Acid Bacteria of Sweet Corn Based Milk

1 1.6 x 107 1.9 x 107 2.2 x 107

10 8.3 x 106 1.2 x 107 1.2 x 107

20 6.2 x 106 1.1 x 107 9.4 x 106

30 5.0 x 106 7.5 x 106 8.2 x 106

40 3.0 x 106 3.9 x 106 2.8 x 106

Source: Kosasih, 2012

3.2.Viability of Lactic Acid Bacteria in Legume and Sweet Corn Based Milk

From Table 5,6,7,8 can be found that the viability of L. plantarum EM1 in soy bean, mung bean, kidney bean, and sweet corn milk were higher compared with L.

pentosus EM 1 from day 1 until day 40 storage. L. plantarum EM1 has higher stability

towards certain conditions such as low pH and high acid. Beside concentrations of total acids and pH, cell viability as affected by many factors especially physico-chemical properties and processing parameters, include the strains used, interaction between species present, culture conditions, the fermentation medium (carbohydrate source), hydrogen peroxide content due to bacterial metabolism, availability of nutrients, growth promoters and inhibitors, dissolved oxygen incubation temperature, fermentation time and storage temperature (Rajiv I. Dave, 1998).

Table 5. Viability of Lactic Acid Bacteria in Soy Bean Based Milk

Day Viability of Lactic Acid Bacteria in Soy Bean Based Milk (%)

10 90.35 86.68 86.69

20 87.42 85.73 85.75

30 80.22 79.55 73.38

40 73.75 65.16 58.89

Source: Yani, 2012

Table 6. Viability of Lactic Acid Bacteria in Mung Bean Based Milk

Day Viability of Lactic Acid Bacteria in Mung Bean Based Milk (%)

L.bulgaricus L.plantarum EM1 L.pentosus EM1

10 98.76 95.37 96.72

20 93.12 93.74 93.72

30 83.63 86.26 90.03

40 81.84 84.35 77.87

Source: Dewi, 2012

Table 7. Viability of Lactic Acid Bacteria in Kidney Bean Based Milk

10 96,34 96,77 91,73

20 94,04 93,95 90,51

30 90,65 91,53 88,35

40 88,21 87,10 67,75

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6 Table 8. Viability of Lactic Acid Bacteria in Sweet Corn Based Milk

10 96,24 97,52 96,32

20 94,44 96,70 94,96

30 93,18 94,64 94,14

40 90,13 90,65 87,87

Source: Kosasih, 2012

3.3. Biochemical Changes of Probiotik Drink

Lactic acid is one of major product of sugar degradation due to the bacterial fermentation via glycolysis pathway while lactic and acetic acids via pentose phosphate pathway. Figure 1, 2, 3, 4 shows the changes of total acidity and total sugar content in all probiotic drink samples during period of storage. The initial content of total acid ranges from 0.27-0.62% while the final total acid content ranges from 0.36-0.62% for all samples. This final value of total acid have met the regulation for fermented milk probiotik (minimal 0.3%) based on CODEX, 2003. The highest total acid content up to is showed by Lactobacillus plantarum EM1 inoculated in kidney bean probiotic drink after 40 days storage.

Figure 1-4. Total Acid and Sugar Content of Legumes and Sweet Corn Probiotic Drink

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7 Table 9. pH and Protein Content Legumes and Sweet Corn Probiotic Drink for 40 Days Storage

Charac- teristic

Source L. bulgaricus L. plantarum EM1 L. pentosus EM1 Initial After 40

days

Initial After 40 days

pH Soy bean 3,97 ± 0,01 3,98 ± 0,01 3,95 ± 0,01 4,03 ± 0,00 3,98 ± 0,01 4,00 ± 0,00 Mung bean 4,03  0,01 4,03  0,00 4,45  0,01 4,20  0,00 4,06  0,01 3,86  0,00 Kidney bean 4,06 ± 0,00 4,02 ± 0,00 4,07 ± 0,01 4,00 ± 0,00 4,02 ± 0,00 3,99 ± 0,00 Sweet Corn 4,13 ± 0,02 4,11± 0,00 4,29 ± 0,00 4,24± 0,00 4,17 ± 0,00 4,10 ± 0,00 Protein

Content (%)

Soy bean 2,75 ± 0,04 2,16 ± 0,02 2,13 ± 0,02 1,97 ± 0,02 2,23 ± 0,02 2,12 ± 0,01 Mung bean 2,96  0,03 2,73  0,04 3,00  0,08 2,80  0,10 3,02  0,25 2,84  0,02 Kidney bean 2,07 ± 0,02 2,24 ± 0,01 2,09 ± 0,03 2,22 ± 0,04 2,02 ± 0,01 2,16 ± 0,02 Sweet Corn 3,21 ± 0,02 2,85 ± 0,01 3,35 ± 0,16 2,75 ± 0,01 3,17 ± 0,03 2,72 ± 0,12 Proteolysis is required by LAB for growth and also acid production (Thomas & Pritchard, 1987 in Maurad & Meriem, 2008). In this study, two strains have shown good proteolysis in soybean, mungbean and sweetcorn probiotic drink (Table 9). But after 40 days storage in refrigerator, only mungbean and sweetcorn probiotic drink have met the regulation of fermented probiotik drink (minimal 2,7%) according to Codex, 2003.

4. CONCLUSION AND FUTURE STUDY

From the result of total bacteria and viability of Lactic Acid Bateria in legume and sweet corn based milk, can be seen that both bacteria (L. plantarum EM1 and L.

pentosus EM1) have potential to be used as inculum or starter in legume based milk to

produce probiotic beverage. Sweet corn inoculated with Lactobacillus plantarum EM1 after 40 days storage has the best characteristics as a probiotic based on total LAB, cell viability, and other biochemical characteristics such as pH, total acid and protein content. Moreover, future study is needed to evaluate the sensory aspects of legumes and sweet corn based milk which made using these two bacteria as inoculum and to evaluate the survival of Lactobacillus plantarum EM1 and Lactobacillus penthosus

EM1 in probiotic drink in simulated gastrointestinal conditions.

ACKNOWLEDGEMENT

We would like to thank Dr. Lindayani, MP and Dra. Laksmi Hartayanie, MP (Lecturer in Under Graduate and Post Graduate Program of Food Technology), as our advisors. And for Margaretha Evelyne (Post Graduate Program of Food Technology), Citra Dewi, Tabitha Yani, Amelia Kosasih and Elizabeth Ria (Under Graduate Program of Food Technology) for the permission to use the data to complete the paper.

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