Wine Production from Papaya Piece Using Immobilized Yeast (Saccharomyces cerevisiae) and Its Physicochemical Analysis

 

C. Maragatham1 and A. Panneerselvam2*

1PG and Research Dept. of Microbiology, PRIST University, Thanjavur-614 904, Tamil Nadu, India.   

2PG Dept. of Botany and Microbiology, A.V.V.M Sri Pushpam College, Thanjavur-613503, Tamil Nadu, India.

Corresponding Author E-mail: maragathamalagesan@yahoo.com

 

ABSTRACT:

Suitability of papaya pieces for the preparation of immobilized yeast biocatalyst for wine production was investigated by using Saccharomyces cerevisiae in repeated batch fermentation. The fermentation rate and other parameters were compared with free yeast cells at different temperatures. The biocatalyst, which was optimized and adapted to low temperature (15ºC) used for the continuous fermentation. The yeast was bound on both surface and with in the pieces. Continuous fermentation was carried out for 40 days. The system was operated at both 15 and 20ºC. The papaya supported biocatalyst was stable in both mechanically and physically during fermentation. There are no changes in cell metabolism of immobilized yeast. Preliminary sensory tests established the fruity, fine taste and the overall improved quality of the produced wine.

 

KEYWORDS: Wine production, immobilization, repeated batch fermentation, papaya pieces.

 

 


INTRODUCTION:

Production of wine is a very old finding because of the natural character of the product. It is defined as an alcoholic beverage obtained from grape juice using yeast as a fermenting organism (1). In the mid of 19th century application of immobilized enzymes were developed and the application of this technique was introduced in the field of analysis and industrial production of biomolecules (2). In wine making procedure also brought many changes from its traditional methods with the progress of biotechnology (3). In recent years, cell immobilization techniques have become increasingly important and are being successfully applied in industrial process such as the production of alcohols, organic acids, enzymes and biotransformation of steroids for hormone production, waste water treatment, food applications, beer, wine production (4-6). Production of wine using immobilized yeast in repeated batch fermentation and high cell density reactors have several advantages over the traditional fermentation by free cells, such as increase in productivity and reduction in cost of product. The host for yeast immobilization for wine making is always biologically inert (7).

 

Many investigations proposed many supports for immobilization for yeast cells like alginate and other inorganic materials. In alcohol fermentations there is a wide variety of supports are used by many investigators (4,5,8). But in wine fermentation the immobilization in low cost is the big problem because the alginate and organic materials are proved inappropriate for wine production and they are not in use. Bardi and Koutinas (9) have been reported the delignified cellulosic material for wine fermentation.

 

In this situation the fruits are the alternative for supports in wine fermentations. Previously few investigators were done good piece of work in this field they used apple pieces, grape skins and quince and pear pieces as solid supports wine production in repeated batch and continuous processes (5,9,10,11,12). In the present study the suitability of papaya pieces as support for wine fermentation by Saccharomyces cerevisiae was investigated. The biocatalyst prepared by using papaya fruits was used in batch fermentation using papaya fruits.

 

MATERIALS AND METHODS:

Yeast strain:

Saccharomyces cerevisiae culture isolated from rotten papaya fruits. The culture was maintained on malt agar medium and stored at refrigerator.

 

Inoculum preparation:

The inoculum was prepared by inoculating the slant culture into 10ml test tube incubated overnight then 3% inoculum transferred to 100ml of the sterile malt broth medium taken in 250ml flask and grown it on a rotary shaker for 48 h. After incubation period the culture was used for preparation of wine.

 

Preparation of papaya must:

Papaya variety Co2 was procured from Tamil Nadu Agricultural University, Coimbatore (India). Papaya fruits were completely peeled off. The pulp was macerated in mixie/blender and pasteurized at 85-90ºC for 5 minutes. After cooling the pulp required amount of cane sugar was added to adjust the final TSS to 24ºBrix. The must was used without any nutrient addition and adjustment.

 

Yeast cell immobilization:

The method used for preparation of guava pieces biocatalyst was previously described (6).  Two hundred gram of papaya fruits were made into 2-3 cm pieces sterilized 121ºC for 15 min. These pieces were taken into a wide mouth 500ml conical flask then 200ml of culture medium contain yeast cell optical density of 2 at 590 nm. pH was adjusted to 5 and left for 8-12 h for fermentation. The fermented broth was decanted to remove the un immobilized yeast cell. The biocatalyst prepared by this method was used for the repetitive batch fermentation and continuous fermentation. In batch fermentation the biocatalyst was washed twice with 200ml of papaya must after all fermentation.

 

For the determination of yeast cell number 10g of wet papaya pieces were taken from the conical flask during fermentation. The pieces are homogenized with mechanical mortar and pestle 90 ml of ¼ strength Ringer solution(13). The cells were counted after appropriate dilution by using a Neubauer improved heamocytometer. 3 x 106 yeast cells gm-1 of papaya fruit pieces were attached.

 

Fermentation:

Repeated batch fermentations were carried out by 200g of biocatalyst prepared as above were used to ferment 400ml of papaya must. The biocatalyst was kept submerged in case of attach fermentation by means of plastic netting. The fermentation was conducted at various temperatures like 15, 20, 25, 30 and 35ºC. The end points of fermentation are detected with measuring of residual sugar less than 2%.

 

Methods of analysis:

The total sugar was determined as per the procedure described(14). The alcohol content was determined as per the standard procedures(15)

 

Organoleptic evaluation:

The sensory evaluation was done using 8 judge panels after aging for 1 month. Observations were recorded for color, clarity, body and taste on a 5 point scale with 5 points for excellent quality and 1 point for bad quality.

 

RESULT AND DISCUSSION:

The prepared biocatalyst was used in about 15 repeated fermentations. Every time the biocatalyst was washed and used. The yeast strain used had the advantage of alcohol tolerance. The fermentation results are summarized in Table-1. All the fermentations were carried out using papaya supported biocatalyst with same initial concentration of sugar (20% w/v). The obtained wine contained alcohol at concentrations similar to dry and table wines (9.5 - 12% v/v). The fermentations carried out continuously for 100 days without any significant loss of the biocatalitic activity. At 10ºC the fermentations were completed in 4 days while at 30ºC it takes only 36 h. Both wine and ethanol productivities were some fold higher than in traditional fermentations and were significantly affected by temperature (p>0.05), at low temperatures (15ºC), an improvement of fermentation time and productivity was observed. This may probably due to adaptation of the immobilized cells (Table1).

 

Among the immobilized yeast cell the wine and ethanol productivity was slightly reduced after first three-repeated fermentations. This may be due to difficulty in nutrient transfer, since a decrease in the papaya biocatalyst was observed and therefore, yeast cells were not uniformly spread throughout the whole mass of must. Therefore, the first and second batches were carried out with 300 and 250ml, respectively and the subsequent batch with 200ml. This decrease was probably due to the utilization of the papaya sugar by the yeast cells. The papaya pieces volume remained stable after seventh or eighth batch. There is no decrease in volume until the end of fermentations, which was mainly due to the residual cellulosic content of papaya an unfermentable constituent. Although there was significant loss of papaya constituents to the wines, pretreatment was not required. Because, most papaya constituents, glucose, fructose, organic acids, phenolic compounds and others which are generally present in red wine. This may also contribute to the flavor and distinctive aroma of the product and therefore it might be useful. The viability of yeast cells was high in immobilized cells. Since the cell number in the immobilized cells increased as a result of yeast cells are detached from support and grew in the medium solution, which initially no yeast cells were preset in the media(16). The appearance of yeast cells in the medium occurs after 12 h of fermentation. Free cell biomass concentrations for the duration of the experiment at all temperatures ranged from 0.3 to12.8 gl-1. But the cell mass in the immobilized pieces are maintained constantly. It may be due to the new cells were absorbed by the support (10). Both the total and volatile acidities of produced wine was similar to table and dry wines.

 

Variation in fermentation performance:

The rate of fermentation was increased in low temperature (15ºC) and this in turn is decreased the time that required for fermentation. The sugars were completely converted to ethanol. Biocatalyst was produced higher amount of ethanol than free cells. Glycerol may be high in immobilized cell medium due to the immobilized cells subjected some stress in addition to this the biocatalyst gave low percent of sugars to medium. This may also contribute for formation of high amount of ethanol by biocatalyst.

 


Table1: Fermentation parameters obtained in repeated batch fermentations at different temperature.

Temp(ºC)

Repeated batches

Initial sugar (%)

Fermentation time (h)

Ethanol concentration % (v/v)

Reducing sugar (%)

Total acidity (%)

30( C )

3

21.0

72

12.0

0.73

0.94

30

1

20.2

72

11.0

0.68

0.96

30

2

20.0

35

12.0

0.53

0.83

30

3

20.1

24

12.0

0.53

0.98

25( C )

3

20.0

100

10.0

0.54

1.20

25

4

20.2

70

10.0

0.33

0.99

25

5

20.5

55

10.5

0.56

1.22

25

6

20.0

48

10.2

0.33

1.13

20(C)

3

20.0

120

9.5

0.65

0.99

20

7

20.3

83

10.5

0.59

1.98

20

8

20.1

65

10.0

0.54

0.97

20

9

20.6

42

10.4

0.28

1.01

15( C)

3

20.2

180

9.3

0.73

0.94

15

10

20.4

120

11.2

0.68

0.96

15

11

20.2

90

10.9

0.53

0.84

15

12

20.0

96

10.0

0.53

0.982

C = Control with free yeast cell.

 


Continuous fermentation:

Continuous fermentation was carried out to investigate the operational stability and suitability of the immobilized yeast strain Saccharomyces cerevisiae on papaya pieces in this work. The biocatalyst, which was optimized and adapted to low temperature (15ºC) used for the continuous fermentation. The yeast was bound on both surface and with in the pieces. Continuous fermentation was carried out for 40 days. The system was operated at both 15 and 20ºC. The papaya supported biocatalyst was stable in both mechanically and physically during fermentation.

 

Sensory evaluation:

The sensory evaluation was done using 8 judge panels after aging for 100 days. Observations were recorded for color, clarity, body and taste on a 5 point scale with 5 points for excellent quality and 1 point for bad quality.  The papaya wine fetched an overall quality score of 4.6 for ripe stage and 3-4.3 for remaining stages. The results were compared with local commercial wine and free cell wine. The preliminary study characterized the new wine as a novel, special type of wine, with a pleasant, color, clarity, body, and fruity taste (Table: 2).

 

Table 2: Sensory evaluation results of papaya wine obtained by the free and immobilized yeast cells (5 point scale)

TREATMENT

Color

Clarity

Body

Taste

Wine from free cell

3.72

3.61

3.28

4.05

Immobilized on papaya pieces

3.83

3.72

3.83

4.68

 

CONCLUSIONS:

With the results obtained in the present study it can be concluded that the mixed effects of temperature and immobilization produces wines with better ratio of esters to alcohols which finally gives more fruity character to the produced wine. Papaya immobilized yeast biocatalyst contributes as a good and effective support for alcoholic fermentation both at low and room temperatures. They are cheap, readily available of food grade and there is no need to special pretreatment before their use. The immobilization on papaya pieces not negatively affected the fermentation. The immobilized biocatalyst shows better stability and increases the fermentation rate, particularly at low temperature, which makes its use possible in industries.

 

 

 

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Received on 30.12.2010       Modified on 19.01.2010

Accepted on 06.02.2011      © RJPT All right reserved

Research J. Pharm. and Tech. 4(5): May 2011; Page 798-800