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Item Influence of fermentation on the quality of Fijian Theobroma cacao beans over two harvest seasons(Taylor and Francis Group, 2024-10-01) Raju RN; Heyes J; Archer R; Chen QTheobroma cacao beans are cultivated in Fiji by smallholder farms in low volumes, mainly for export. Cocoa beans are often processed under ambient conditions by farmers and cocoa processors. Postharvest processing includes natural fermentation from six to ten days in wooden boxes, followed by sun – drying in the open for up to 14 days. The impact of fermentation conditions on key quality parameters of Fijian cocoa beans, such as temperature profile, pH, and total extractable polyphenol content (TPC) are presented in this study. The quality of fermentation was assessed using a standard method, such as a cut test followed by fermentation index (FI) measurement. A temperature increase to 40°C and variations in the pH of the bean mass were evident during natural fermentation. TPC in the cocoa beans was sensitive to temperature and pH. Fermentation was variable due to weather conditions between harvest seasons. The dry season had the best conditions for fermentation as the peak temperature of the bean mass was 40°C and the FI was high (1.39 ± 0.04) There is a need to improve fermentation during the rainy season. This can be achieved by providing training to Fijian cocoa farmers on using better fermentation techniques.Item Response of Ruminal Microbiota-Host Gene Interaction to High-Altitude Environments in Tibetan Sheep.(MDPI (Basel, Switzerland), 2022-10-17) Sha Y; Ren Y; Zhao S; He Y; Guo X; Pu X; Li W; Liu X; Wang J; Li S; Wahli WAltitude is the main external environmental pressure affecting the production performance of Tibetan sheep, and the adaptive evolution of many years has formed a certain response mechanism. However, there are few reports on the response of ruminal microbiota and host genomes of Tibetan sheep to high-altitude environments. Here, we conducted an integrated analysis of volatile fatty acids (VFAs), microbial diversity (16S rRNA), epithelial morphology, and epithelial transcriptome in the rumen of Tibetan sheep at different altitudes to understand the changes in ruminal microbiota−host interaction in response to high altitude. The differences in the nutritional quality of forage at different altitudes, especially the differences in fiber content (ADF/NDF), led to changes in rumen VFAs of Tibetan sheep, in which the A/P value (acetic acid/propionic acid) was significantly decreased (p < 0.05). In addition, the concentrations of IgA and IgG in Middle-altitude (MA) and High-altitude Tibetan sheep (HA) were significantly increased (p < 0.05), while the concentrations of IgM were significantly increased in MA (p < 0.05). Morphological results showed that the width of the rumen papilla and the thickness of the basal layer increased significantly in HA Tibetan sheep (p < 0.05). The 16S rRNA analysis found that the rumen microbial diversity of Tibetan sheep gradually decreased with increasing altitude, and there were some differences in phylum- and genus-level microbes at the three altitudes. RDA analysis found that the abundance of the Rikenellaceae RC9 gut group and the Ruminococcaceae NK4A214 group increased with altitudes. Furthermore, a functional analysis of the KEGG microbial database found the “lipid metabolism” function of HA Tibetan sheep to be significantly enriched. WGCNA revealed that five gene modules were enriched in “energy production and conversion”, “lipid transport and metabolism”, and “defense mechanisms”, and cooperated with microbiota to regulate rumen fermentation and epithelial immune barrier function, so as to improve the metabolism and immune level of Tibetan sheep at high altitude.Item Transcriptomic and proteomic changes associated with cobalamin-dependent propionate production by the rumen bacterium Xylanibacter ruminicola.(American Society for Microbiology, 2024-10-29) Mahoney-Kurpe SC; Palevich N; Gagic D; Biggs PJ; Reid PM; Altshuler I; Pope PB; Attwood GT; Moon CDXylanibacter ruminicola is an abundant rumen bacterium that produces propionate in a cobalamin (vitamin B12)-dependent manner via the succinate pathway. However, the extent to which this occurs across ruminal Xylanibacter and closely related bacteria, and the effect of cobalamin supplementation on the expression of propionate pathway genes and enzymes has yet to be investigated. To assess this, we screened 14 strains and found that almost all strains produced propionate when supplemented with cobalamin. X. ruminicola KHP1 was selected for further study, including complete genome sequencing, and comparative transcriptomics and proteomics of KHP1 cultures grown with and without supplemented cobalamin. The complete KHP1 genome was searched for cobalamin-binding riboswitches and four were predicted, though none were closely located to any of the succinate pathway genes, which were dispersed at numerous genomic loci. Cobalamin supplementation led to the differential expression of 17.5% of genes, including genes encoding the cobalamin-dependent methylmalonyl-CoA mutase and some methylmalonyl-CoA decarboxylase subunits, but most propionate biosynthesis pathway genes were not differentially expressed. The effect of cobalamin supplementation on the KHP1 proteome was much less pronounced, with the only differentially abundant propionate pathway enzyme being methylmalonyl-CoA mutase, which had greater abundance when supplemented with cobalamin. Our results demonstrate that cobalamin supplementation does not result in induction of the entire propionate biosynthesis pathway, but consistently increased expression of methylmalonyl-CoA mutase at transcriptome and proteome levels. The magnitude of the differential expression of propionate pathway genes observed was minor compared to that of genes proximate to predicted cobalamin riboswitches. IMPORTANCE In ruminants, the rumen microbial community plays a critical role in nutrition through the fermentation of feed to provide vital energy substrates for the host animal. Propionate is a major rumen fermentation end-product and increasing its production is desirable given its importance in host glucose production and impact on greenhouse gas production. Vitamin B12 (cobalamin) can induce propionate production in the prominent rumen bacterium Xylanibacter ruminicola, but it is not fully understood how cobalamin regulates propionate pathway activity. Contrary to expectation, we found that cobalamin supplementation had little effect on propionate pathway expression at transcriptome and proteome levels, with minor upregulation of genes encoding the cobalamin-dependent enzyme of the pathway. These findings provide new insights into factors that regulate propionate production and suggest that cobalamin-dependent propionate production by X. ruminicola is controlled post-translationally.Item Control of citrinin and pigments produced by Monascus purpureus during the fermentation of red fermented rice : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatu Campus, New Zealand(Massey University, 2024) Abdul Halim, FarawahidaMonascus spp. is a fungal starter used to produce red fermented rice (RFR). This product has some health benefits and contains pigments that can act as natural colour and flavouring agents. However, Monascus spp. can also produce the mycotoxin, citrinin (CIT) which is believed to have adverse effects on human health. CIT in RFR has been reported worldwide by using different methods of detection. In this current research, Coconut Cream Agar (CCA) was developed as a simple and rapid semiquantitative method to screen CIT–producing Monascus spp. isolates from RFR. Two Monascus purpureus isolates, MF1 and MS1, were isolated. These isolates were identified based on the macroscopic and microscopic observations, and deoxyribonucleic acid (DNA) sequencing [internal transcribed spacer (ITS) and beta–tubulin (β–tubulin) genes]. Further analysis showed that these isolates contained polyketide synthase (pksCT) and ctnA genes, which are CIT biosynthesis genes. These isolates exhibited fluorescence on CCA and were confirmed as CIT producers by Ultra–high performance liquid chromatography with a fluorescence detector (UHPLC–FLD). A toxicity test was conducted using Artemia salina to determine the toxicity of CIT. The results showed that LC50 was 66 µg/mL. The fungal growth, CIT, pigments production, and pH of M. purpureus isolates were characterized on CCA for 30 days. Decreasing CIT levels were observed after incubation of MF1 and MS1 on CCA for 8 and 7 days, respectively. The pigments increased during this incubation time. There were similar trends for CIT and pigments observed during the production of RFR. CIT increased to a maximum level after 5 days of incubation and pigments increased from 5–9 days. There appears to be a relationship between pigments production and CIT levels during the growth of M. purpureus. Mixing CIT and pigments extracted from MF1 and MS1 resulted in a reduction of CIT by 26–68% and 16–45%, respectively. The results on specific pigments and their effect on CIT showed that ankaflavin, monascin, monascorubrin, rubropunctatin, and monascorubramine significantly reduced CIT, with the highest reduction produced by ankaflavin. The findings of this study suggest pigments production could be optimized to control CIT levels in Monascus–fermented products.Item The Effects of Unfermented and Fermented Cow and Sheep Milk on the Gut Microbiota.(Frontiers Media S.A., 2019-03-06) Rettedal EA; Altermann E; Roy NC; Dalziel JE; Mayo BA variety of fermented foods have been linked to improved human health, but their impacts on the gut microbiome have not been well characterized. Dairy products are one of the most popular fermented foods and are commonly consumed worldwide. One area we currently lack data on is how the process of fermentation changes the gut microbiota upon digestion. What is even less well characterized are the possible differences between cow and other mammals' milks. Our aim was to compare the impact of unfermented skim milk and fermented skim milk products (milk/yogurt) originating from two species (cow/sheep) on the gut microbiome using a rat model. Male Sprague-Dawley rats were fed a dairy-free diet supplemented with one of four treatment dairy drinks (cow milk, cow yogurt, sheep milk, sheep yogurt) for 2 weeks. The viable starter culture bacteria in the yogurts were depleted in this study to reduce their potential influence on gut bacterial communities. At the end of the study, cecal samples were collected and the bacterial community profiles determined via 16S rRNA high-throughput sequencing. Fermentation status drove the composition of the bacterial communities to a greater extent than their animal origin. While overall community alpha diversity did not change among treatment groups, the abundance of a number of taxa differed. The cow milk supplemented treatment group was distinct, with a higher intragroup variability and a distinctive taxonomic composition. Collinsella aerofaciens was of particularly high abundance (9%) for this group. Taxa such as Firmicutes and Lactobacillus were found in higher abundance in communities of rats fed with milk, while Proteobacteria, Bacteroidetes, and Parabacteroides were higher in yogurt fed rats. Collinsella was also found to be of higher abundance in both milk (vs. yogurt) and cows (vs. sheep). This research provides new insight into the effects of unfermented vs. fermented milk (yogurt) and animal origin on gut microbial composition in a healthy host. A number of differences in taxonomic abundance between treatment groups were observed. Most were associated with the effects of fermentation, but others the origin species, or in the case of cow milk, unique to the treatment group. Future studies focusing on understanding microbial metabolism and interactions, should help unravel what drives these differences.Item Characteristisation of acetic acid bacteria and yeast isolated from kombucha produced in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Auckland, New Zealand. EMBARGOED until 21 August 2026.(Massey University, 2024-08-13) Wang, BoyingBackground: Kombucha is a popular functional tea beverage commonly fermented by a complex symbiotic culture of acetic acid bacteria (AAB) and yeast (SCOBY) in a base of sugared tea infusion at ambient temperature for 7-14 days. Regular consumption of Kombucha confers potential health benefits due to the presence of live cultures and high concentrations of bioactive components such as vitamins, polyphenols, and organic acids. However, industrial production of Kombucha faces challenges due to the limited information on the dynamic changes in its microbial community composition and the lack of knowledge regarding their health-promoting characteristics. The impact of fermentation conditions and added substrates on starter cultures, physicochemical characteristics and functional activities is also not well understood. Objectives: This study aimed to determine the microbiological characteristics of New Zealand Kombucha starter cultures and evaluate the probiotic potential of AAB and yeast isolated from commercial Kombucha products. The bioactive components, antioxidant activity, and antimicrobial activities of Kombucha fermented using a New Zealand starter culture under different fermentation conditions were also determined.--Shortened abstractItem Extracellular Polysaccharide Extraction from Streptococcus thermophilus in Fermented Milk.(American Society for Microbiology, 2022-04-27) Wa Y; Chanyi RM; Nguyen HTH; Gu R; Day L; Altermann E; Cocolin LLactic acid bacteria such as Streptococcus thermophilus are known to produce extracellular polysaccharide (EPS) in fermented foods that enhance the creaminess and mouthfeel of the product, such as yogurt. Strains producing larger amounts of EPS are highly sought-after, and therefore, robust and accurate quantification methodologies are important. This study found that two commonly used methodologies significantly underestimated the amount of EPS produced as measured using a milk matrix. To this end, a proteolytic step was implemented prior to EPS extraction (Method C). An initial proteolytic step using xanthan gum-spiked milk significantly increased recovery yield to 64%, compared to 27.8% for Method A and 34.3% for Method B. Method C showed no improvement when assessed using a chemically defined medium. Method C was further validated using three strains of S. thermophilus with varying EPS-production capabilities (STLOW, STMID, STHIGH). Overall, Method C demonstrated significant improvements in the EPS extraction yield for all three S. thermophilus strains in fermented milk. On average, Method C improved isolation yield by ∼3- to 6-fold compared with Method A and by ∼2- to 3-fold compared with method B. There were no significant differences between samples when they were grown in a chemically defined medium, highlighting the importance of a proteolytic step specifically for fermented milk samples. In commercial applications, accurate quantification of EPS-production is an important aspect when finding new strains. IMPORTANCE Extracellular polysaccharide (EPS) production by milk-fermenting microorganisms is a highly sought-after trait in improving the perceived thickness, creaminess, and mouthfeel of yogurt. Streptococcus thermophilus are commonly isolated and their EPS production is quantified in the search for higher-producing strains. In this study, we demonstrated that two commonly used methods for isolating EPS from milk samples significantly underestimated the true amount of EPS present. We demonstrated that the addition of a proteolytic step prior to EPS extraction isolated over 2-fold more EPS than identical samples processed using the traditional protocols. We further validated this method in fermented milk samples from three strains of S. thermophilus that included a low-, mid-, and high-EPS producing strain. Again, we showed significant improvements in EPS isolation using a proteolytic step. In the search for new S. thermophilus strains with enhanced EPS production, accurate quantification in an optimal medium is essential.Item Effect of Divergent Feeding Regimes During Early Life on the Rumen Microbiota in Calves(Frontiers Media S.A., 2021-10-20) Cristobal-Carballo O; McCoard SA; Cookson AL; Laven RA; Ganesh S; Lewis SJ; Muetzel S; Morgavi DPThe objective of this study was to determine whether divergent feeding regimes during the first 41 weeks of the life of a calf are associated with long-term changes in the rumen microbiota and the associated fermentation end-products. Twenty-four calves (9 ± 5 days of age) were arranged in a 2 × 2 factorial design with two divergent treatments across three dietary phases. In phase 1 (P01), calves were offered a low-milk volume/concentrate starter diet with early weaning (CO) or high-milk volume/pasture diet and late weaning (FO). In phase 2 (P02), calves from both groups were randomly allocated to either high-quality (HQ) or low-quality (LQ) pasture grazing groups. In phase 3 (P03), calves were randomly allocated to one of two grazing groups and offered the same pasture-only diet. During each dietary phase, methane (CH4) and hydrogen (H2) emissions and dry matter intake (DMI) were measured in respiration chambers, and rumen samples for the evaluation of microbiota and short-chain fatty acid (SCFA) characterizations were collected. In P01, CO calves had a higher solid feed intake but a lower CH4 yield (yCH4) and acetate:propionate ratio (A:P) compared with FO calves. The ruminal bacterial community had lower proportions of cellulolytic bacteria in CO than FO calves. The archaeal community was dominated by Methanobrevibacter boviskoreani in CO calves and by Mbb. gottschalkii in FO calves. These differences, however, did not persist into P02. Calves offered HQ pastures had greater DMI and lower A:P ratio than calves offered LQ pastures, but yCH4 was similar between groups. The cellulolytic bacteria had lower proportions in HQ than LQ calves. In all groups, the archaeal community was dominated by Mbb. gottschalkii. No treatment interactions were observed in P02. In P03, all calves had similar DMI, CH4 and H2 emissions, SCFA proportions, and microbial compositions, and no interactions with previous treatments were observed. These results indicate that the rumen microbiota and associated fermentation end-products are driven by the diet consumed at the time of sampling and that previous dietary interventions do not lead to a detectable long-term microbial imprint or changes in rumen function.Item In Vitro Assessment of Hydrolysed Collagen Fermentation Using Domestic Cat (Felis catus) Faecal Inocula(MDPI (Basel, Switzerland), 2022-02-17) Butowski CF; Thomas DG; Cave NJ; Bermingham EN; Rosendale DI; Hea S-Y; Stoklosinski HM; Young W; Ebani VVThe gastrointestinal microbiome has a range of roles in the host, including the production of beneficial fermentation end products such as butyrate, which are typically associated with fermentation of plant fibres. However, domestic cats are obligate carnivores and do not require carbohydrates. It has been hypothesised that in the wild, collagenous parts of prey-the so-called animal-derived fermentable substrates (ADFS) such as tendons and cartilage-may be fermented by the cat's gastrointestinal microbiome. However, little research has been conducted on ADFS in the domestic cat. Faecal inoculum was obtained from domestic cats either consuming a high carbohydrate (protein:fat:carbohydrate ratio of 35:20:28 (% dry matter basis)) or high protein (protein:fat:carbohydrate ratio of 75:19:1 (% dry matter basis)) diet. ADFS (hydrolysed collagen, cat hair, and cartilage) were used in a series of static in vitro digestions and fermentations. Concentrations of organic acids and ammonia were measured after 24 h of fermentation, and the culture community of microbes was characterised. The type of inoculum used affected the fermentation profile produced by the ADFS. Butyrate concentrations were highest when hydrolysed collagen was fermented with high protein inoculum (p < 0.05). In contrast, butyrate was not detectable when hydrolysed collagen was fermented in high carbohydrate inoculum (p < 0.05). The microbiome of the domestic cat may be able to ferment ADFS to provide beneficial concentrations of butyrate.Item Kombucha: Production and Microbiological Research(MDPI (Basel, Switzerland), 2022-10-31) Wang B; Rutherfurd-Markwick K; Zhang X-X; Mutukumira ANKombucha is a sparkling sugared tea commonly prepared using a sugared tea infusion and fermented at ambient temperature for several days using a cellulose pellicle also called tea fungus that is comprised of acetic acid bacteria and yeast. Consumption of Kombucha has been reported as early as 220 B.C. with various reported potential health benefits and appealing sensory properties. During Kombucha fermentation, sucrose is hydrolysed by yeast cells into fructose and glucose, which are then metabolised to ethanol. The ethanol is then oxidised by acetic acid bacteria (AAB) to produce acetic acid which is responsible for the reduction of the pH and also contributes to the sour taste of Kombucha. Characterisation of the AAB and yeast in the Kombucha starter culture can provide a better understanding of the fermentation process. This knowledge can potentially aid in the production of higher quality products as these microorganisms affect the production of metabolites such as organic acids which are associated with potential health benefits, as well as sensory properties. This review presents recent advances in the isolation, enumeration, biochemical characteristics, conventional phenotypic identification system, and modern genetic identification techniques of AAB and yeast present in Kombucha to gain a better understanding of the microbial diversity of the beverage.
