Traditional Inoculum Research Articles

Genotypic Characterization of Rhizopus species from Tempeh and Usar: Traditional Inoculum of Tempeh in Indonesia

Abstract. Soybeans tempeh (tempeh) is processed by fermentation using Rhizopus spp. Tempeh is an important source of protein in Indonesia. The traditional inoculum in fermentation locally is known as Usar which is made from the leaves of Hibiscus tiliaceus. However, Rhizopus information from Usar is still limited. Therefore, this study aims to identify and investigate the genetic diversity of Rhizopus species from Usar and tempeh based on the Internal Transcribed Spacer (ITS) sequence and the Random Amplified Polymorphic DNA (RAPD) markers. Twenty-three Rhizopus strains were isolated from Usar and ten Rhizopus strains were isolated from tempeh. Based on ITS sequences, the isolates were similar to R Rhizopus microsporus (30 isolates) and Rhizopus delemar (3 isolates) with 98-99% similarity. The genetics of R. microsporus and R. delemar are varied and different from the genetics of R. microsporus from tempeh. The growth temperature of R. microsporus varies from 33 to 48°C and R. delemar can grow to a maximum at 33°C. The role of R. microsporus and R. delemar from Usar in determining the quality of tempeh is still limited. Therefore, it needs to be investigated further.

Production Methods and Physicochemical Characteristics of Cassava Inoculum and Attiéké from Southern Côte d’Ivoire

Attiéké is a food from Côte d’Ivoire exported today in several countries. For evaluating production processes, determinant factors and quality attributes of attiéké, a production survey and a physicochemical study were carried out. The survey included 170 producers from the departments of Abidjan, Dabou and Jacqueville, major production areas. Traditional attiéké (Adjoukrou, Ebrié, Alladjan) and a commercial type Garba were investigated for physicochemical analyses. The cassava variety (98% of producers) had no effect on traditional cassava inoculum but had an impact on attiéké quality. The step of fermentation is necessary. The difference between studied attiéké related to microflora of inoculum whose identification may provide adequate explanations on the product. Respect for the various steps of manufacturing process is also essential. Physicochemicals confirmed observed differences between attiéké types. Organoleptic characteristics are criteria of differentiation between attiéké. The quality of preference is well-made grains without fibers. The results obtained also highlighted the risks faced by regular consumers of Garba due to its high cyanide content (12 mg/100g MS) compare to other attiéké (4.41 mg/100g MS).

Indonesian millennial consumers’ perception of tempe – And how it is affected by product information and consumer psychographic traits

Nine different tempe (five traditional and four modernised versions of tempe) were tested by 165 millennial consumers (63.6% women) in blind and informed sessions. The effects of information on hedonic response, sensory perception, collative properties and the product-elicited emotions were evaluated. The provided information related to the raw materials (bean type), origin (local or imported) and the production methods (traditional inoculum usar, with starter culture inoculum ragi and use of stainless steel factory processing equipment hygienis). The millennial consumers’ hedonic response and other parts of their perception were highly affected by the product information. The five traditional tempe were more liked when the product information was provided. In addition, significantly differences in collative properties were observed when product information is provided. Particularly the collative properties Authentic and Traditional. The elicited emotion that were most affected were Proudness. Surprisingly, a number of sensory properties were also affected significantly by the provided information. However, the interaction is not systematic neither for bean nor production type. Segmentation of respondents based on their level of Food Neophobia and Attitudes Towards Traditional Foods (ATTF), showed profound differences. ATTF segmentation in particular, as it affected both hedonic response, elicited emotions, as well as sensory and collative properties. Pro-traditional consumers reacted more positively to the information than other segments. The results demonstrated the powerful effect of transparency regarding the products’ provenience and product methods. The results show the importance of the millennial consumers’ education and information to preserve or maintain the diversity of food we eat.

Formation of ACE-inhibitory peptides during fermentation of jack bean tempe inoculated by usar Hibiscus tiliaceus leaves starter

Jack bean is one of the potential legumes as a source of ACE-inhibitory peptides in tempe fermentation due to its high protein content and amino acids composition. A traditional inoculum for tempe fermentation is usar from Hibiscus tiliaceus leaves. The aim of this study was to investigate the optimum tempe fermentation time inoculated using usar on the formation of ACE-inhibitory peptides. The study applied the prolonged tempe fermentation (120 h) at room temperature (+30°C). The change of pH, soluble protein, protease activity, and ACE-inhibitory activity during tempe fermentation were determined using standard methods. There were significant differences (α=0.05) in pH, proteolytic activity, soluble protein content, and ACE-inhibitory activity during tempe fermentation. The highest protease activity was produced at 96 h of fermentation while the highest soluble protein content was produced at 120 h of fermentation with the value of 12.71 unit/g and 3.65 mg/mL, respectively. The highest ACE-inhibitory activity was reached at 72 h of fermentation (53.89 %). Conclusion: The optimum fermentation time of the formation of ACE-inhibitory peptide was at 72 h of fermentation.

Genetic diversity of Rhizopus microsporus from traditional inoculum of tempeh in Indonesia based on ITS sequences and RAPD marker

Abstract. Barus T, Halim R, Hartanti AT, Saputra PK. 2019. Genetic diversity of Rhizopus microsporus from traditional inoculum of tempeh in Indonesia based on ITS sequences and RAPD marker. Biodiversitas 20: 847-852. The main microorganism for tempeh fermentation is Rhizopus microsporus. These days, many tempeh producers use commercial inoculum, such as ‘Raprima’ as resource of R. microsporus. As a result, the genetic diversity of R. microsporus that had been reported in Indonesia has diminished. Information about genetic diversity is needed as a basis to select R. microsporus as tempeh inoculum. This research aims to investigate the genetic diversity of R. microsporus from waru leaves based on Internal Transcribed Spacer (ITS) Sequence and Random Amplified Polymorphic DNA (RAPD) markers. A total of 25 R. microsporus were isolated from traditional inoculum waru leaves (Inoculum 1) and traditional inoculum other than waru leaves (Inoculum 2). Amplification of ITS sequence was done using universal primer pairs of ITS-4 and ITS-5. Amplification of RAPD markers was done using primers OPC-08, OPC-19, OPQ-6, R-108, OPA-09 and OPJ-20. ITS sequence was not sufficient to compare the similarities among R. microsporus. On the other hand, RAPD markers successfully compared the similarities among 25 R. microsporus. A total of 25 R. microsporus were divided into 9 clusters. R. microsporus from Inoculum 1 grouped into Cluster 1, Cluster 3 and Cluster 4-8. Inoculum 2 grouped into Cluster 2 and Cluster 9. R. microsporus from tempeh grouped into Cluster 4 and was different from Inoculum 1 and Inoculum 2, except for TB3.

Isolasi dan identifikasi kapang Rhizopus pada tempe gude (Cajanus cajan L.)

Penelitian yang telah dilakukan ini bertujuan untuk mengisolasi kapang Rhizopus pada tempe gude, seperti diketahui bahwa Rhizopus adalah kapang yang sangat penting dalam proses fermentasi tempe. Penelitian ini diawali dengan pembuatan tempe menggunakan biji gude sebagai substrat, yang diinokulasi dengan inokulum tradisional (usar), diinkubasi pada temperatur kamar sampai menjadi tempe (48-72 jam). Kapang tempe gude diisolasi dan diidentifikasi dengan menggunakan metode pengenceran berseri dan spread plate. Hasil penelitian menunjukkan bahwa biji gude sebagai substrat pertumbuhan Rhizopus sp. Kapang tempe gude didominasi oleh kapang berwarna putih keabu-abuan dan putih abu-abu kecoklatan yang diidentifikasi mirip dengan Rhizopus oligosporus.

Sensory Profiling and Hedonic Evaluation of Attiéké from Local and Improved Cassava Varieties

Attieke is fermented cassava semolina steamed and consumed with proteins and vegetables. From Ivorian traditional origin, its popularity is increasing in Africa and production networks are developing. However, the growth of attieke industry is still curbed by lack of technical skills of producers and irregular quality, depending on cassava variety, processing and inoculum. In the present study, a traditional inoculum and four cassava varieties (two improved local (IAC and Bonoua) and two improved sweet ones from Nigeria (Olekanga and TMS 4 (2) 1425)) were used for producing attieke Ebrie. For the sensorial evaluation of prepared attieke, qualitative sensory profiling was used. The sensory quality descriptors where quantitatively measured by a trained sensory panel. With this method the most important quality criteria of attieke were judged. These quality criteria were selected with the aid of surveys on its production and consumption. The relationships between sensory attributes of the four attieke prepared of the different cassava varieties and biochemical characteristics were studied. Sensory analysis revealed that the four attieke were cream-colored products with sweet and/or sour tastes, made of cohesive and well-formed grains of different sizes, with an odor of fermentation specific to attieke and showing a firm texture. The differences between sensory profiles of the four attieke produced from four cassava varieties (12 months growth) concern only some descriptors of quality. Pearson correlation coefficients between physicochemical descriptors of quality showed that biochemical parameters may help predict organoleptic characteristics of attieke Ebrie (pH-size of grains: r = 0.99; pH-acidity: r = −0.92; acidity-rounded grains: r = −0.98: starch-cohesion between grains: r = −0.96; starch-sweet: r = −0.95; starch-granulous: r = 0.97, total sugar-yellow color: r = 0.96; total sugar-aroma of attieke: r = 0.96; reducing sugar-odor of attieke: r = 0.95; cyanide-fibrous: r = 0.95).

Influence of microbiological and chemical quality of traditional starter made from cassava on “attiéké” produced from four cassava varieties

Attiéké is a steamed fermented cassava semolina and food now included in many African countries habits. Non-control of production factors is an important constraint causing manufacturing defaults. Moreover, toxic varieties are used in fermented foods.The aim of this study was to characterize and optimize microbiological, physicochemical and biochemical qualities of attiéké Ebrié. The analyses were performed on traditional inoculum and 4 cassava varieties.The lactic fermentation of cassava dough had positive impact on microbiological and chemical qualities of prepared attiéké that showed slight acidity (pH = 4.6), high starch (80–90 g/l00 g) and low cyanide contents (2.79–5.00 mg/kg). Therefore, sweet varieties are suitable for preparing attiéké.

In vitro study of probiotic properties of Lactobacillus plantarum F22 isolated from chhang - A traditional fermented beverage of Himachal Pradesh, India.

Present study was carried out to evaluate a new bacterial strain, Lactobacillus plantarum F22 as probiotic strain. L. plantarum F22 was isolated from a traditional inoculum called ‘Phab’ which is used for the preparation of a traditional beverage chhang of Lahaul and Spiti of Himachal Pradesh. The isolate was identified by conventional and molecular techniques and tested for different probiotic properties. The 16S rRNA sequence of the isolate was registered in National Centre for Biotechnology Information (NCBI) under accession number KT865223. Further, L. plantarum F22 was evaluated for its probiotic potential viz., autoaggregation capacity, hydrophobicity, acidity tolerance, antibiotic susceptibility and cumulative probiotic potential and was found to possess good probiotic potential with a cumulative probiotic score of 91.7%. L. plantarum F22 has been proved to be highly effective, therefore can be recommended for the development of new pharmaceuticals and functional food preparations.

Technology of Preparation and Consumption Pattern of Traditional Alcoholic Beverage ‘Sur’ of Himachal Pradesh

A survey of ‘Sur’ production in Himachal Pradesh (India) was conducted and reported. Sur is a traditional alcoholic beverage produced in many parts of Himachal using finger millet (Eleucine corcana) as a basic raw material. A crisp review of the survey revealed that the Sur is known by various local names in the different regions. Knowledge regarding preparation and consumption pattern of Sur was collected by carrying out a survey in Kullu, Kangra, Mandi and Sirmaur districts of Himachal Pradesh. The fermentation is carried out mostly in the earthen pots and these were sterilized by the smoke produced by burning jaggery and ‘dhaeli’ inside the pots and retaining the smoke for some time. Its fermentation is carried out by using a traditional inoculum dhaeli which is a mixture of roasted barley with several herbs with natural micro-flora. It is made from barley, weighed from 250 gm to 1 Kg, yellow in color and need 1–2 to months for preparation. The fermentation is generally carried out in the traditional earthen wares which takes about 7–8 days in summers and 12–15 days in winters to complete. The method of preparation though varied in different districts surveyed. Based on the information collected, a general flow sheet for the process was developed and reported here. The product also has social and religious significance. It is offered to the local deities and is served in the social gatherings like marriages, funerals and other religious ceremonies. As it makes use of the several herbs it is also supposed to have medicinal properties. It is usually consumed in the un-distilled form. The beverage is regarded as a tonic by the elders and is most popular among them. However, a decreased trend of sur consumption was found among the youngsters as they had shifted to other commercially available beverages in the market.

Biochar characteristics relate to its utility as an alternative soil inoculum carrier to peat and vermiculite

Biochar materials have greatly variable physical and chemical properties, which will affect their abilities to serve as carriers for introducing bacteria into soils. Here we examined biochars made from 5 feedstocks produced at 2 highest treatment temperatures (HTT's) by slow pyrolysis. Peat and vermiculite, which are traditional inoculum carriers, and liquid inoculum with no carrier, were included for comparison. All of the carriers were inoculated with a liquid suspension of the plant growth promoting rhizobacterial (PGPR) strain, Enterobacter cloacae UW5, carrying a green fluorescent protein (GFP) marker. Inoculum survival was determined using quantitative PCR to enumerate the GFP markers in DNA extracted from non-sterilized soils directly after incorporation of the inoculated carriers and after a 4-week incubation. The biochars were characterized with respect to carbon and nitrogen content, specific surface area, pH, electrical conductivity, water holding capacity, pore opening diameters, and hydrophobicity to identify specific attributes that influence the survival of the inoculant after introduction into soil. The results indicated that chemical properties of biochar, particularly nitrogen and pH, were among the most important characteristics affecting initial inoculum survival and hence likely the shelf life. However, once incorporated into soil, physical features, including surface area, pore opening diameters, and water-filled pore spaces, were more closely associated with inoculum survival. All biochars tested performed as well as vermiculite and none demonstrated detrimental effects on the UW5 population. The best biochar was that made from pinewood at a HTT of 600 °C (Pine600), which performed as well as peat and sustained higher population densities than vermiculite. The Pine600 biochar was further tested to assess its effect on the expression of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and auxin synthesis, both of which were unaffected by the presence of biochar at 2% or 5% (w/v) concentrations.

Influence of gaseous VOC concentration on the diversity and biodegradation performance of microbial communities

In this work, the influence of toluene gas concentration on the isolation of toluene degrading microbial communities from activated sludge was studied. Toluene biodegradation at gas phase concentration of 10 g m(-3) (R1) resulted in process instability with removal efficiencies (RE) lesser than 33 %, while operation at toluene gas phase concentrations of 300 mg m(-3) (R2) and 11 mg m(-3) (R3) was stable with RE ranging from 74 to 94 %. The consortium isolated in R1 exhibited the highest tolerance toward toluene but the lowest biodegradation performance at trace level VOC concentrations. Despite R2 and R3 showed a similar sensitivity toward toluene toxicity, the microbial community from R2 supported the most efficient toluene biodegradation at trace level VOC concentrations. The Shannon-Wiener index showed an initial biodiversity decrease from 3.2 to 2.0, 1.9 and 2.7 in R1, R2 and R3, respectively. However, while R2 and R3 were able to recover their initial diversity levels by day 48, this loss in diversity was permanent in R1. These results showed that traditional inoculum isolation/acclimation techniques based on the exposure of the inoculum to high VOC concentrations, where toxicity tolerance plays a key role, may result in a poor abatement performance when the off-gas stream is diluted.

Screening for mycotoxins in the inoculum used for production of attiéké, a traditional Ivorian cassava product

For the production of attiéké, a fermented staple food from the Ivory Coast, a traditional inoculum consisting of mould-covered cassava tubers is used. Nineteen inoculum and five attiéké samples of different provenance were tested for contamination with the major mycotoxins ochratoxin (OTA), aflatoxin (AF) B 1, B 2, G 1, G 2 and deoxynivalenol (DON). Selected samples were also subjected to an effect-based bioassay visualising noxious impact on a test organism, the bacterium Vibrio fischeri. Neither AF nor DON were detected and only trace amounts of up to 0.2 μg/kg OTA were present in some samples, but extract fractions inhibiting the biological test system were discovered through the bioassay. Our findings suggest that the most potent fungal toxins do not present a general health hazard to attiéké consumers. Unidentified bioactive microbial metabolites, however, might still be present and affect the human or animal organism in an unknown manner.

Characterisation of the microflora of attiéké, a fermented cassava product, during traditional small-scale preparation

Attiéké is a fermented cassava product consumed mainly in Cote d'Ivoire. The aim of this study was to characterise the attiéké fermentation by examining products from 15 small-scale production sites at various stages of its preparation. For the preparation of attiéké, fresh cassava is grated to a pulp and inoculated with 10% of a spontaneous traditional inoculum. The inocula contained aerobic mesophiles at mean numbers of 8.2 × 10 7 cfu/g and lactic and acetic acids at mean concentrations of 0.2% and 0.1%, respectively. The mean pH was 5.0. Lactic acid bacteria were the dominant microorganisms in cassava pulp throughout fermentation with the mean numbers being 1.2 × 10 9 cfu/g after 15 h. The identification to the species level of microorganisms from one representative attiéké production of good quality showed that, at the start of fermentation, Leuconostoc mesenteroides subsp. mesenteroides was present in the highest numbers, accounting for 20% of all lactic acid bacteria. As the fermentation proceeded, this species was replaced by homofermentative lactic acid bacteria, Lactobacillus salivarius and Lactobacillus delbrueckii subsp. delbrueckii, present at 20% and 16%, respectively, and obligate heterofermentatives, Lactobacillus fermentum and Lactobacillus confusus at 12% and 10%, respectively, of total lactic acid bacteria in the flora at the end of fermentation. High numbers of acid-sensitive microorganisms, Bacillus circulans, Bacillus lentus, Enterobacter sakazakii, Enterobacter cloacae and Klebsiella pneumoniae subsp . pneumoniae, were transferred to the pulp in the inocula, but acidification to a mean pH of 4.4 with mean lactic and acetic acid concentrations of 0.59% and 0.2%, respectively, prevented their growth and reduced their numbers to less than 10 2 cfu/g at the end of fermentation. The mean numbers of Candida tropicalis, the main yeast present, remained relatively constant at about 10 5 cfu/g throughout attiéké production. The mean numbers of aerobic mesophiles decreased to below 10 2 cfu/g as a result of the steaming process. The finished attiéké had a mean pH of 4.4 and mean lactic and acetic acid concentrations of 0.6% and 0.1%, respectively.

PENGARUH FERMENTASI TERHADAP KANDUNGAN PROTEIN DAN KOMPOSISI ASAM AMINO DALAM SINGKONG

Effects of Solid Fermentation on Protein Content and Amino Acid Composition of Cassava. This study was carried out to assess the protein and amino acid quantity of solid fermentation of cassava (Manihot esculenta) using pure culture of the Rhizopus oligosporus and traditional inoculum (laru). The protein content of the fermented product was analyzed by Biuret method, and the animo acid composition by HPLC (high performance liquid chromatography) method. The results showed that solid fermentation of cassava increased the protein content from 2.1% to 4.0% and 4.7%. The animo acid contents of the fermented product increased 2.5 folds of that of cassava. Higher increase was seen in substrates fermented with traditional inoculum. This is due to the addition of coconut oil and ammonium to the cassava substrate which improved the growth of mold. Key words: cassava (Manihot esculenta), food fermentation, protein content, amino acid composition

Effect of cassava variety and processing methods on the performance of ‘kudeme’ in agbelima production

Kudeme, a traditional inoculum used in fermenting cassava for the production of agbelima may be prepared from different cassava varieties and by different processing methods. The effects of these two variables on the performance of kudeme were studied using three cassava varieties and three processing methods. Performance was measured as a function of the ability of kudeme to improve texture, colour and flavour of agbelima. Both cassava varietal difference and processing method affected significantly ( p < 0.01) the performance of kudeme. The ‘Bosomensia’ variety of cassava and the ‘Blanching’ method for the processing of kudeme gave the best results. The interaction between the two variables also significantly ( p < 0.05) affected the performance of the kudeme. The cassava variety/processing method combinations which gave the best performing kudeme samples were the Blanched Bosomensia (BBN), Blanched Biafra (BBF) and the Toasted Bosomensia (TBN) in descending order of performance. These results serve as a useful guide in selecting cassava varieties and processing methods for preparing kudeme and constitute a significant step in optimizing the production process of agbelima.

Hibiscusleaves for the manufacture of usar, a traditional inoculum for tempe

AbstractTempe is an Indonesian proteinaecous food produced from soya beans by fermentation mainly byRhizopus spp. A common traditional inoculum for this fermentation is usar, consisting of heavily sporulatedRhizopusspp of which the mycelium, grown on added and inoculated soya beans, adheres to leaves of the waru tree (Hibiscusspp). A description of various waru trees is given together with their suitability for usar making. The epiphytic fungi on waru trees of various geographical origins (Indonesia, Africa, The Netherlands) were investigated.Cladosporiumspp were present on all the leaves sampled. However,Rhizopusspp predominated over the other fungi encountered on the Indonesian leaves. Experimental incubation of leaves with and without added soya beans showed that the epiphytic fungi are less competitive thanRhizopusspp and will not interfere if normal usar‐making conditions are observed. Experiments with alternative attachment surfaces (paper, a textile) indicated that the waru leaves do not provide environmental selectivity through growth‐inhibiting substances, but that they serve mainly as a convenient attachment surface with moisture‐retaining capacity while providing adequate aeration for fungal development.

Cassava detoxication during tapé fermentation with traditional inoculum

SummaryTapé ketela is a traditional Indonesian fermented cassava product using ragi as inoculum. Cassava fermented with 0.2–0.4% ragi produced a characteristic flavour and sweet‐sour alcoholic taste. After 3 days' fermentation the pH decreased from 6.54 to 5.06–5.17, titratable acidity increased from 0.07 to 0.22–0.26%, reducing and total sugars increased from 1.1 to 21.4–22.0% and 3.9 to 22.9–23.5%, respectively, while alcohol content increased from less than 0.01 to 2.02–2.12%. Total cyanide loss during fermentation was 58–87%. Most cyanide was lost during steaming (40–70%) while 9–35% was lost during 3 days fermentation. Sensory evaluation revealed no significant differences in flavour, texture and colour (P &gt; 0.01) of tapé prepared by two different methods. Tapé prepared from dried cassava (gaplek) did not develop characteristic organoleptic properties.

Phytic acid changes in soybeans fermented by traditional inoculum and six strains of Rhizopus oligosporus

Tempeh was prepared from Delmar variety soybeans inoculated with the traditional Indonesian inoculum (usar) and six pure culture strains of Rhizopus oligosporus. The strains BTU3K1 and CT11K2 produced the best quality tempeh. The phytic acid content of soybeans was reduced from 1mD07% in whole dry soybeans to 0mD67–0mD75% in tempeh. Most mould strains did not have a significantly different effect on reducing the phytic acid content in tempeh, although the traditional inoculum was significantly different to strains CTU7K2, BTU3K1, BT1K4 and CT11K2.