Lactic Acid Bacteria, Fatty Acid Profile, and Quality of Cocoghurt During Fermentation

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Abstract

Cocoghurt is a novel fermentation product produced coconut milk as the main raw ingredient. In this study, the effect of starter concentration and fermentation time on viability of lactic acid bacteria (LAB), as well as the fatty acid profile and quality of cocoghurt was examined. Lactobacillus casei subsp. casei R-68 and Streptococcus thermophilus were used as starter cultures.

The study was carried out using a completely randomized design with variations in fermentation time. The data was statistically analyzed using ANOVA and DNMRT at 5%.

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Parameters quantified were total LAB, pH, total lactic acid, total solids, protein, moisture and ash content as well as total fat and fatty acid profile.

The results show that 3.0% of Lactobacillus casei subsp. casei R-68 and Streptococcus thermophilus starter resulted in optimal growth of LAB. Fermentation time significantly affected pH, total lactic acid, total LAB and protein content, but did not significantly inluence, ash, moisture, fat and total solid content.The duration of fermentation also did not significantly affect fatty acid profile. Probiotic cocoghurt fatty acid profiles consisted mainly of medium-chain saturated fatty acids followed by long-chain saturated fatty acids and finally unsaturated fatty acids.

Introduction

Indonesia is one of the largest coconut producers in the world, with production estimated at 18.3 million tons per year in various regions. Riau Province is the largest coconut-producing area in Indonesia with a planting area of 520,260 Ha from a total of 3,654,520 Ha; coconut production was reported at 427,080 tons in 2015 and 418,250 tons in 2016 (Indonesian Central Bureau of Statistics, 2017). Coconut milk is one of the main products from raw coconut processing.

However, incorporation of coconut milk in local snacks is still very limited. Cocoghurt is one type of fermented product from coconut milk which has the potential to be developed.

The importance of fermentation in modern-day life is underlined by the wide spectrum of foods marketed both in developing and industrialized countries, not only for the benefit of preservation and safety, but also for their highly appreciated sensory attributes. Fermented foods are treasured as major dietary constituents in numerous developing countries because of their keeping quality under ambient conditions - thereby contributing to food security - and because they add value, enhance nutritional quality and digestibility, improve food safety, and are traditionally acceptable and accessible (Holzapfel, 2002; Rolle and Satin, 2002). Recently, there is a growing interest to develop a variety of fermented products for other beneficial purposes, particularly for health purposes and preventing of toxins produced by foodborne pathogens and spoilage bacteria that enter human body (Shah, 2007; Ali, 2010).

Lactobacillus casei subsp. casei R-68 (LCR-68) was isolated from dadih, a fermented food produced from local buffalo milk originating from West Sumatra, Indonesia (Hosono et al., 1989). Fermented cocoghurt is produced using commercial starter S. thermophilus and the local LAB, LCR-68. The two starter cultures were expected to optimize the production and quality of cocoghurt. LCR-68 was selected as starter due to its toleranance to stomach acid and bile salts, allowing it to survive in the digestive tract when consumed. This strain has several benefits, including lowering blood cholesterol, acting as an antimutagen and preventing cancer.

Furthermore, previous reports indicate its ability to attact invading pathogenic bacteria such as Escherichia coli, Listeria monocytogeneses, and Staphylococcus aureus in vitro (Pato et al., 2017a) and in vivo (Pato et al ., 2017b). The release of anti-microbial compounds by beneficial LAB into the gut results in inactivation of pathogenic bacteria, balances the intestinal microflora, prevents infection and reduces pathogenesis (Remacle and Reusens, 2004). According to Sreekumar and Hasono (2000), LAB is able to reduce production of carcinogens in the gut by suppressing the growth of pathogenic bacteria that convert procarcinogenic compounds into carcinogens via the activity of the enzymes β-glucuronidase and β-glucosidase. The activity of this enzyme is strongly related to the total lactic acid bacteria present in the intestine. Hence, total LAB can be used as a marker in cancer prevention, especially colon cancer.

Fermented foods and beverages may vary based on the nature of the food, the fermentation time, amount of initial starters, and the intentional application of microbes utilized (Anal, 2019). In yogurt production, S. thermophilus lowers the pH by metabolizing lactose in milk into lactate and lactic acid, which triggers the growth of L. bulgaricus (Tamime and Deeth, 1980). Based on this role that S. thermophilus plays in yogurt fermentation, we hypothesize that addition of S. thermophilus to coconut milk fermentation would trigger the growth of LCR-68 to produce cocoghurt.

There are several factors that affect the final quality of fermentation products, one of which is the length of fermentation. Fermentation time affects bacterial activity because the longer the fermentation time, the more active the bacteria and the higher the total LAB; as a result, increased amounts of substrate in the media is metabolized (Surono, 2004). Duration of fermentation is one of the most important factors affecting the physical, chemical and organoleptic properties of fermented processed products (Kunaepah, 2008). Previous findings show that that 15 hours of fermentation using LCR-68 as a starter resulted in probiotic fermented milk (Pato et al., 2015) and 6 hours of fermentation using Enterococcus faecalis UP-11 resulted in cocoghurt (Iman et al., 2015) that meets the Indonesian standard of fermented milk (SNI 10-2981-2009).

Since coconut milk is the main ingredient in cocoghurt, the food product contains medium fatty acids which may be beneficial to health. The major MCFA (medium chain fatty acid) present in coconut milk is lauric acid which has many health benefits such as increasing immunity as well as being an antimicrobial agent (Lieberman et al., 2006; Nakatsuji et al., 2009; Shilling et al., 2013; Dayrit, 2014; Anzaku et al., 2017). Until now there have been no findings that report the fate of the MFCA during fermentation. Therefore, the objective of the present study was to evaluate the effect of starter concentration and fermentation time on the viability of lactic acid bacteria, fat profile and the quality of cocoghurt.

Materials and Methods

The study was carried out experimentally using a completely randomized design for determining the effects of starter concentration and fermentation time on cocoghurt quality. For the first experiment, starter concentration was varied as follows: C1 (starter concentration 1.5%), C2 (starter concentration 3.0%), C3 (starter concentration 4.5%), C4 (starter concentration 6.0%) and C5 (starter concentration 7.5%). In the second experiment, fermentation time was varied as follows: T1 (fermentation time for 6 h), T2 (fermentation time for 10 hours), T3 (fermentation time for 14 hours), T4 (fermentation time for 18 hours) and T4 (fermentation time for 22 hours). Each treatment was repeated 3 times.

Lactobacillus casei subsp. casei R-68 (LCR-68) isolated from dadih by Hosono et al (1989) and commercial Streptococcus thermophillus were used as bacterial starters in this study.

Coconut milk was prepared according to Pato et al. (2019). Grated coconut was squeezed using a coconut milk press until the coconut milk was completely extracted from the grated coconut. The coconut milk was then filtered using a filter cloth, and pure coconut milk was obtained.

The starter used was prepared in two stages; the first stage involved preparation of medium containing 5% skim milk and 2% sucrose. This medium was stirred evenly, then transferred into a glass jar and sterilized at 121ºC for 10 minutes. Following cooling down to 30-40ºC, the skim milk medium was inoculated with LCR-68 and S. thermophylus separately (2% v/v) and incubated at 37ºC for 13 hours. Next, a second medium was prepared consisting of equal volumes of skim milk and coconut milk. The mix was stirred evenly, transferred into glass jars and sterilized at 121ºC for 10 minutes. After cooling down to 30-40ºC, the skim milk/coconut milk mix was inoculated with bacteria from the first skim milk medium. The second medium was used as the active starter for making cocoghurt.

The cocoghurt was prepared following formulation previously described by Pato et al., (2019). Coconut milk (400 ml) was mixed with sucrose 2% (v/w), skim milk 5% (v/w) and carboxymethyl cellulose 0.05%. The mixture was then homogenized using a blender for 5 minutes. The homogenized media was heated to 85°C for 15 minutes, then cooled to a temperature of 37°C. The media was then inoculated with starter LCR-68 and S. thermophilus at concentrations ranging between 1.5 to 7.5%, then incubated at 37°C for 6, 10, 14, 18 and 22 hours as described.

The cocoghurt parameters measured were pH, total lactic acid, total LAB, protein, fat, total solids, moisture and ash content. The methods used were in accordance with Indonesian National Yoghurt Standard (Indonesia National Standardization Agency, 2009). pH was determined using a pH meter, while protein, ash, total solids, moisture and fat content were analyzed according to the method described by Sudarmadji et al., (1997). Total lactic acid was determined by alkalimetric titration using 0.1N NaOH, and total LAB was determined according to methods described by Fardiaz (1998). Fatty acid profile was analyzed using gas chromatography (Seppanen-Laakso et al., 2002).

The data obtained was analyzed using analysis of variance (ANOVA). For test results with F count greater than or equal to F table, further testing was performed using the Duncan New Multiple Range Test (DNMRT) at 5% to determine the differences between each treatment.

Results and Discussion

Optimal LAB growth occurred at 3.0% starter concentration. Fermentation time significantly altered pH, lactic acid, and protein levels, while not affecting other quality parameters. The fatty acid profile predominantly featured medium-chain saturated fatty acids. This study underscores the importance of starter concentration and fermentation time in enhancing cocoghurt's microbial and nutritional quality.

Conclusion

Cocoghurt presents a promising fermented product from coconut milk, with L. casei subsp. casei R-68 and S. thermophilus effectively improving its viability and fatty acid profile. Adjusting starter concentration and fermentation duration can optimize cocoghurt's health benefits and quality.