The Effect of Inoculum Types On The Yeast Growth Pattern During Tempe Fermentation Samsul Rizal

  

The Effect of Inoculum Types On The Yeast Growth Pattern

During Tempe Fermentation

1) 1) 1) 1)

  

Samsul Rizal , Maria Erna Kustyawati , Murhadi , Udin Hasanudin , and

2) Fatimah 1)

  

Lecturers of Department of Agricultural Product Technology, Faculty of

  Agriculture, University of Lampung

  2)

  Graduated of Department of Agricultural Product Technology, Faculty of Agriculture, University of Lampung

  Email: marrizal @yahoo.com

  

ABSTRACT

Tempe is a food made of soybeans fermentation and inoculated with Rhizopus

oligosporus in solid fermentation. Besides R. oligosporus, other microorganisms

such as bacteria and yeasts were found during fermentation. This study aimed to

know the effect of inoculum types on the pattern of yeast growth during tempe

fermentation with addition of Saccharomyces cerevisiae. The research was done

by Randomized Complete Block Design (RCBD) with two factors and three

replications. The first factor was the types of tempe inoculum, consist of 4 levels,

i.e. commercial inoculum, Saccharomyces cerevisiae, Rhizopus oligosporus, and

mixture of Rhizopus oligosporus and Saccharomyces cerevisiae. The second

factor was fermentation time, consist of 6 levels, i.e. 0, 8, 16, 24, 32, and 40

hours. The results showed that yeast grew during the soybean fermentation with

commercial tempe inoculum, Saccharomyces cerevisiae, and mixture of Rhizopus

oligosporus and Saccharomyces cerevisiae, but they didn’t grow during soybean

fermentation with addition of R. oligosporus only. After lag phase until 8 hours

of fermentation time, the yeast growth increased until the end of fermentation,

although it decreased at 32 hours of fermentation with mixture of Rhizopus

oligosporus and Saccharomyces cerevisiae.

  Keywords: Tempe, yeast, Saccharomyces cerevisiae . Pattern of Growth

  INTRODUCTION

  Tempe is a food made by fermenting soybeans inoculated with Rhizopus

  oligosporus in solid fermentation. The fermentation of tempe is a two-stage

  fermentation, which is fermentation by bacterial activity that takes place during the process of soaking soybeans, and fermentation by mold that takes place after being inoculated with mold (Kustyawati, 2009). R. oligosporus plays a major role in making tempe because it can maintain most of the nutrients contained in soybeans, increase its protein digestibility, and increase levels of several kinds of vitamin B (Muchtadi, 2010 in Mursyid, 2014). In addition to R. oligosporus, during tempe fermentation there are also the presence of other microorganisms such as lactic acid bacteria (LAB) and yeast (Efriwati et al., 2013).

  Yeast participates in tempe fermentation (Feng et al., 2007; Kustyawati, 2009). Kustyawati (2009) states that yeast can grow with indigenous bacteria and R.

  

oligosporus during tempe fermentation. The presence of yeast has been reported

  by Samson et al. (1987) in commercial tempeh in the Netherlands, including Trichosporon beigelii , Clavispora (Candida) lusitaniae, C. maltosa, C.

  intermedia , Yarrowia lipolytica, etc. Among these yeasts, Saccharomyces cerevisiae is a yeast that have the potential to produce tempeh containing beta

  glucans due to its ability to produce beta glucans (Pengkumsri et al., 2017). Beta- glucan is a compound that has a positive effect on body health, especially its ability to increase immunity (Hetlan, et al., 2013). The discovery of yeast in tempe fermentation shows that yeast can grow with R.

  

oligosporus and bacteria during the fermentation process and is possible that the

  yeast has an important role in the fermentation. According to research by Rizal, et al . (2018), which added S. cerevisiae as an inoculum, showed that the S.

  cerevisiae can grow with other microbes and produce tempeh containing beta-

  glucans. Therefore, the deliberate addition of S. cerevisiae in tempe fermentation is expected to produce tempeh containing beta-glucans so that the tempeh produced will have more value due to the beta-glucan content. How is the growth pattern of S. cerevisiae during the tempe fermentation process given the addition of various types of inoculum is one of the problems that need to be studied. In this study, we will study the effect of various types of tempeh inoculum on yeast growth patterns during tempe fermentation.

  MATERIALS AND METHODS Materials

  The materials used in this research include commercial yeast (Raprima trademark), pure culture of Rhizopus oligosporus FNCC 6010 and Saccharomyces

  

cerevisiae FNCC 3012 obtained from the UGM Inter-University Food and

  Nutrition Center, imported soybeans with the trademark Soybean USA no. 1 obtained from Mount Sulah in Bandar Lampung, distilled water, peptone water, physiological salt 0.85%, alcohol 70%, Nutrient Broth (NB), Nutrient Agar (NA), Malt Extract Agar (MEA), Dextrose Agar (PDA), aluminum foil, and cotton.

  Research methods

  This study used a Complete Randomized Block Design (RCBD) with two factors and three replications. The first factor is the type of inoculum tempe, consisting of 4 levels, namely tempe commercial yeast (RAPRIMA trademark), Saccharomyces

  cerevisiae , Rhizopus oligosporus, and mixture of Rhizopus oligosporus +

Saccharomyces cerevisiae . The second factor was fermentation time, consisting of

  6 levels, namely 0 hours, 8 hours, 16 hours, 24 hours, 32 hours, and 40 hours. Parameters observed were yeast growth patterns during the fermentation process 0, 8, 16, 24, 32, and 40 hours . The data obtained were analyzed by variance to determine whether there was influence between treatments. The data was further tested using Orthogonal Polynomial - Orthogonal Comparison (OP-OC) test at the level of 5%.

  Preparation of Saccharomyces cerevisiae culture

Saccharomyces cerevisiae from sloping agar was cultured into Malt Extract Agar

  (MEA) media in a petri dish so that S. cerevisiae was obtained in the form of colonies in the media. The S. cerevisiae colonies were transferred into a test tube containing 9 mL of De Man, Rogosa and Sharpe Broth (MRSB) media, then

  o homogenized using vortex and incubated at 28 C for 48 hours. Furthermore, S. speed of 3000 rpm for 10 minutes. Centrifugation was carried out twice, using sterile distilled water in the second centrifugation process. After that, the number of S. cerevisiae cells was calculated using haemacytometer. The number of S.

  7 cerevisiae was adjusted to 10 cells / gram.

  Preparation of Rhizopus oligosporus culture

Rhizopus oligosporus from sloping agar was cultured into Potato Dextrose Agar

o (PDA) media in a petri dish, then incubated for 5-7 days at 25 C so that R. oligosporus was in the form of a colony. The R.oligosporus colonies were harvested using drygalski by adding 5-10 mL of sterile distilled water.

  Furthermore, R. oligosporus spores were centrifuged at 3000 rpm for 10 minutes. After that, a solid R. oligosporus spore was obtained, then diluted in a diluent solution. The number of R. oligosporus spores in the diluent solution was calculated using haemacytometer and it was adjuted to 105 cells / gram.

  Production of Tempe

  The process of making tempe followed the procedure of Kustyawati (2009). A total of 300 grams of soybeans are soaked in clean water at room temperature for one night, then the skin is removed. Furthermore, they were boiled use clean water with a ratio of 1: 3 (soybeans: water) for 30 minutes, drained and dried until the soybean temperature reaches room temperature. The fermentation stage was done by mixing every 100 grams of boiled soybean with tempeh inoculum according to the treatment. After being mixed, the soybean seeds were inserted in a packing plastic that had been perforated regularly for aeration purposes, then incubated at 32 ° C for 40 hours, observed every 8 hours during fermentation.

  Enumeration of Yeast Amount

  Calculation of yeast count was carried out using the Total Plate Count (TPC) method with Malt Extract Agar (MEA) media following the Lay (1994) method. The calculation of the number of yeasts was carried out at 0, 8, 16, 24, 32, and 40 hours of fermentation time. Each sample was sampled and a series of dilutions from 10-1 to 10-10 were made in duplicate. Sample preparation test follows the

  Kustyawati method (2009). A total of 10 grams of tempe sample were mixed with 90 ml of peptone water, then homogenized. After that, a series of dilutions is made to a certain concentration. Furthermore, yeast cultivation was done using the spread plate method on Malt Extract Agar (MEA) media. Incubation was carried

  o out at 32 C for 24-48 hours.

  RESULTS AND DISCUSSION Growth Pattern of Saccharomyces cerevisiae in Tempe Inoculated with Various Types of Inoculum

  The results of the calculation of the number of Saccharomyces cerevisiae cells during the tempe fermentation process inoculated with various types of inoculums showed that Saccharomyces cerevisiae was able to grow during tempe fermentation with the addition of commercial tempe yeast inoculum, inoculum of

  Saccharomyces cerevisiae , or inoculum mixture of Rhizopus oligosporus and Saccharomyces cerevisiae . On the contrary, there was no growth of

Saccharomyces cerevisiae during tempeh fermentation which was inoculated with

Rhizopus oligosporus only. The highest number of yeast cells was found in

  soybeans which were inoculated with Saccharomyces cerevisiae after 40 hour

  9

  fermentation time, which was 4.82 x 10 CFU/g, while the lowest number of yeast cells was found in tempeh inoculated with Rhizopus oligosporus because there was no yeast growth. The number of Saccharomyces cerevisiae cells during soybean fermentation with the addition of tempe yeast, Saccharomyces cerevisiae,

  

Rhizopus oligosporus , and a mixture of Rhizopus oligosporus and Saccharomyces

cerevisiae are presented in Table 1.

  Table 1. Number of S. cerevisiae cells during soybean fermentation by inoculum of tempeh yeast, S. cerevisiae, R.oligosporus, and mixture of R. oligosporus and

  S. cerevisiae .

  Number of S.cerevisiae cells (CFU/g) on the each fermentation duration Types of Inoculum ₃

  8 ₃

  

16

₃

  24 ₄

  32 ₅

  40 ₅ Tempe yeast 1,05 x 10 1,17 x 10 3,87 x 10 3,93 x 10 3,07 x 10 2,63 x 10 _{7 7 8 9 9 9} S. cerevisiae 1,00 x 10 1,50 x 10 1,73 x 10 3,33 x 10 2,77 x 10 4,82 x 10

• R.oligosporus

  R.oligosporus

• + S. cerevisiae 1,0 x 10 1,43 x 10 1,24 x 10 4,07 x 10 1,31 x 10 3,43 x 10

  The results showed that yeast could grow during tempeh fermentation process using inoculum of tempe yeast (Figure 1). Based on the picture, the adaptation phase of yeast growth started from the beginning of fermentation up to 8 hours of fermentation. Kusmiati et al. (2011) stated that the phase of S. cerevisiae adaptation in the media using carbon sources of glucose ended in fermentation at 4 o'clock. Meanwhile, S. cerevisiae grown on Yeast Nitrogen Base (YNB) media containing 30% glucose showed that the adaptation phase was in the first 6 hours fermentation (Ishmayana et al., 2012). The phase of S. cerevisiae adaptation in this study was longer than the research conducted by Kusmiati et al. (2011) and Ishmayana et al. (2012) due to the absence of additional carbon sources which are the main nutrients needed for the growth of S. cerevisiae. After 8 hours, the growth of S. cerevisiae enters the exponential phase until the 32nd hour, then enters the stationary phase until the 40 hours. The death phase of yeast cell was estimated to occur after fermentation lasting more than 40 hours. The yeast presence in the soybean treatment which is inoculated with tempeh yeast is thought to originate from tempeh yeast used or from the surrounding environment.

  12.0

  11.0 g

  10.0 (Lo

  9.0 st

  8.0 a )

  7.0 /g f ye

  6.0 U o

  5.0 r CF

  4.0 be

  3.0 um

  2.0 N

  1.0

  0.0 Fermentation time (hour) Figure 1. Yeast growth in various types of inoculum during tempe fermentation.

  ■ Soybean + S. cerevisiae ˟ Soybean + R. oligosporus + S. cerevisiae ▲ Soybean + R. oligosporus ♦ Soybean + tempe yeast (Merk RAPRIMA)

  Yeast could grow during the soybean fermentation process which was inoculated with S. cerevisiae eventhough tempeh was not formed. Khamir grew by utilizing existing nutrients in soybean substrate. Kustyawati (2010) stated that almost all foods provide adequate nutrition to support yeast growth. In this research treatment, yeast growth was in the adaptation phase from 0 to 8 hours of fermentation time, then increased in the number of yeast cells (logarithmic phase) until 24 hours of fermentation. After this, yeast enters the stationary phase from 24 hours until 40 hours of fermentation time. The yeast death phase was estimated to occur after fermentation lasting more than 40 hours. Soybeans inoculated with R. oligosporus did not show yeast growth until the fermentation time reach 40 hour. This is because there is no addition of yeast during inoculation. This research is in line with Kustyawati (2009) which states that yeast was not found during tempe fermentation using R. oligosporus as inoculum. Thus, this study revealed that the presence of yeast in tempeh can be found if fermented soybeans are added with yeast. However, Nisa (2016) stated

  WJB (Warung Jambu) during fermentation 24 and 48 hours. Research conducted by Nisa (2016) did not specifically calculate the number of yeasts during tempe fermentation. Efriwati et al. (2013) found the presence of yeast from the beginning of soaking, the end of soaking, the beginning of incubation, mid- incubation, and the end of incubation on tempeh made by tempe EMP (once boiling) and EMP tempe (two boiling). The difference between this study and the research conducted by Efriwati et al. (2013) because this study looked at yeast growth patterns during soybean fermentation which was inoculated with R. oligosporus, while research conducted by Efriwati et al. (2013) observed the presence of yeast in the soybean immersion process and during fermented soybeans which were inoculated with tempeh yeast. This study showed that tempeh which was inoculated with R. oligosporus had no yeast growth, while soybean added with yeast tempeh contained yeast growth. Meanwhile, research conducted by Efriwati et al. (2013) used tempe yeast as an inoculum in making tempeh so that yeast was detected. This indicates that the presence of yeast in soybean added with tempeh yeast comes from the yeast of tempe used or from the environment during fermentation. The growth of S. cerevisiae in tempeh which was inoculated with the mixture of

  

R. oligosporus and S. cerevisiae was in the adaptation phase at fermentation time

  0 until 8 hour. After that, S. cerevisiae growth increased until 24 hours of fermentation (logarithmic phase), then dropped to a population of 8.0 Log CFU/g at the fermentation time reach 32 hours (Figure 2). Furthermore, yeast growth increased after fermentation time reach 40 hours. This growth pattern was in line with the growth pattern of S. boulardi which was inoculated together with R.

  

oligosporus as tempeh inoculum in research conducted by Kustyawati (2009). The

  yeast growth pattern in this treatment was similar to the yeast growth pattern in soybeans inoculated with S. cerevisiae only. This showed that S. cerevisiae utilizes nutrients in soybeans for growth and there was a mutually beneficial symbiosis between R. oligosporus and S. cerevisiae during fermentation. According to Kustyawati (2009), there may be mutually beneficial symbiosis in terms of nutrient availability between R. oligosporus and S. cerevisiae during tempe fermentation.

  Figure 2. Response of yeast growth to the duration of fermentation in each type of inoculum.

  Informations: ● soybean + tempe yeast ▲ soybean + S. cerevisiae soybean + R. oligosporus

  ӿ x soybean + R. oligosporus + S. cerevisiae

  The results of variance analysis showed that the amount of yeast during tempe fermentation was influenced by the type of inoculum and the time of fermentation, and there was an interaction between the two. Further test results showed that the number of yeasts during tempe fermentation with the addition of inoculums of R. oligosporus and S. cerevisiae was significantly different from the tempe yeast inoculum, S. cerevisiae inoculum, and R. oligosporus inoculum. Research conducted by Kustyawati (2009) showed that soybeans for tempe fermentation added with different yeast inoculums had different yeast growth patterns. The results of variance analysis showed that the number of yeasts during tempe fermentation was influenced by the type of inoculum and fermentation time, and there was interaction between. The results of further tests showed that yeast quadratic, but significantly in linear so that linear regression was used to determine the effect of tempeh yeast inoculum and fermentation time on yeast growth (Sugiyono, 2007). Linear regression showed that yeast growth in soybean which was inoculated with tempeh yeast, increased until fermentation took place. Based on linear regression, the equation y = 0.0721x + 2.7441 was obtained. This equation means that an increase in fermentation time every 8 hours can increase the amount of yeast by 7.21%. The results of further tests on the treatment of addition of R. oligosporus as an inoculum in soybean fermentation were not significantly different in quadratic or linear terms, meaning that the addition of R.

  oligosporus during soybean fermentation did not affect yeast growth. This is due

  to the absence of yeast growth in this treatment. Further test results showed that the number of yeasts during tempe fermentation with the addition of inoculums of

  R. oligosporus and S. cerevisiae was significantly different from the tempe yeast

  inoculum, S. cerevisiae inoculum, and R. oligosporus inoculum. Research conducted by Kustyawati (2009) showed that soybean for tempe fermentation added with different yeast inoculums had different yeast growth pattern. The treatment of adding S. cerevisiae as an inoculum in tempe making was significantly different in quadratic terms. This shows that during fermentation, there is an increase and decrease in the number of yeasts. Based on the equation y = -0.0012x2 + 0.1242x + 6.7045, the optimum growth point of yeast was obtained at 51.75 hours with a yeast number of 9.92 CFU / g (Figure 6). Similarly, the addition of R. oligosporus and S. cerevisiae treatments were significantly different quadratically. This quadratic difference is because during fermentation, the number of yeasts has increased, then decreased and experienced an increase again. Regression equation in this treatment is y = -0.0012x2 + 0.0856x + 6.8793, so that the optimum point of yeast growth was obtained at 35.67 hours with the number of yeasts was 8.41 CFU / g (Figure 11). The optimum point of addition of R.

  

oligosporus and S. cerevisiae as inoculums was faster than treatment with addition

  of S. cerevisiae. This is presumably because in the addition of R. oligosporus and

  

S. cerevisiae as inoculums in soybean fermentation, S. cerevisiae uses the

  reshuffle by R.oligosporus as a nutrient so that its growth is faster than treatment with addition of S. cerevisiae. This research is in line with Kustyawati (2009) which states that there may be mutually beneficial symbiosis in terms of nutrient availability between R. oligosporus and S. cerevisiae during tempe fermentation.

  Tempe Appearance Inoculated with Various Types of Inoculum during fermentation.

  Miselia produced by R. oligosporus was formed on the surface of soybeans which were inoculated with a mixture of R. oligosporus and S. cerevisiae with a 24-hour fermentation period (Figure 3). Start from 24 to 32 hour of fermentation time, mycelia partially covered the surface of the soybean, while at the fermentation time reach 40 hours, mycelia covered the entire surface of the soybean. The appearance of mycelia is similar to mycelia in the treatment of soybeans which are inoculated using R. oligosporus, which is grayish white and there is a black color. The black color of this tempe was R. oligosporus spore. Spores grew because of the oxygen entering through holes in plastic packaging. According to Bintari et al. (2008), tempe mold is microaerophilic, which requires enough oxygen to grow. Black color decreases in fermentation at the 40th hour, presumably because at the 40th hour of fermentation the spores have formed mycelia which is increasingly compacted so that it covers the surface of the soybean and shows a white appearance.

  

0 jam 8 jam 16 jam 24 jam 32 jam 40 jam

  v Figure 3. Soybeans inoculated with mixture inoculum of R.oligosporus and

  S.cerevisae

  CONCLUSION

  The conclusion of this study is that yeast growth in tempe with the addition of

  

Saccharomyces cerevisiae has increased until the end of fermentation, although it

nd

  had decreased in the 32 hour of fermentation time. Further research is needed to see the yeast growth in tempeh with the addition of Saccharomyces cerevisiae which is added with a carbon source (flour or sugar).

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