Biochemical characterization and In vitro fermentation of bakery waste as an alternative energy source for ruminant feeding

Introduction

By 2050, the global demand for animal products will rise by 60-70% (Derbyshire, 2022; Kırkpınar et al., 2025) [4,16] along with reduction in conventional cereal grain resources (Kilama et al., 2023) [15] . Simultaneously, global food waste is a major part of the socioeconomic and ecological crisis (Soria et al., 2025) [30] , with substantially contribution to greenhouse gas emissions and excessive landfill burdens (Soria et al., 2025) [30] . While cereal grains are highly palatable, energy-dense, and rich in starch (Humer and Zebeli, 2017) [12] , their intensive use is associated with metabolic and ruminal fermentation disorders in highyielding ruminants. Consequently, exploring alternative, non-human-edible by-products like bakery waste, noodle waste, and snack processing leftovers offers a viable strategy for waste disposal and resource recycling (Narasimmalu and Ramasamy, 2020) [20] . From a regulatory and sustainability perspective, utilizing these industrial streams in animal nutrition helps valorize food waste within a circular bioeconomy framework (Nath et al., 2023; Shurson et al., 2023; Bytyqi et al., 2025) [2,21,29] . Bakery waste is highly energy-dense ingredient characterized by higher amount of digestible non-fibrous carbohydrate, gelatinized starch and lipid; and provide a cost-effective substitute for cereal grains like barley and maize (Nayohan et al., 2024; Obeidat, 2025) [22,24] . So, use of industrial by-product, mainly bakery waste provide sustainable circular economy strategy to reduce feed wastage and environmental pollution with alternative to grain (Pinotti et al., 2021) [26] . However, inclusion of bakery by-products in ruminant feed is limited by variation in biochemical composition, processing effects (i.e., time and temperature) and effective inclusion level of bakery waste (Pastorelli et al., 2023; Obeidat, 2025) [24,25] . [27,28] along with gross energy (Bomb Calorimeter) as well as aflatoxin level (Aflatoxin analyzer). [31] NE L (Mcal/kg) = 0.0245 × TDN (%) -0.12 (NRC, 2001; Marchi et al., 2013) [17] The moisture-free samples were sent to Punjab Biotechnology Incubator (PBTI), Laboratory (NABL accredited), Mohali-160059 and were analyzed for starch, sugar, major minerals, micro-minerals, heavy metals, rancidity and microbial parameters as presented in Table 1.

In Vitro gas production and fermentation analysis

For in-vitro analysis, nine dietary levels were prepared with bakery waste inclusion as the substitute for cereal grain at 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35% and 40%, while the level of grain was proportionally reduced from 35% to 0%. Ingredient composition of the concentrate mixture containing different levels of Bakery waste is presented in Table 2.

The nutritional value of wastes was assessed by in vitro gas production (IVGP) technique described by Menke and Steingass (1988). For this trial, rumen liquor was collected from fistulated bulls fed on conventional diet consisting of roughage and concentrate to meet their requirement as per NRC (2001), before feeding at 09:00 h in a thermos flask flushed with CO 2 and maintained at 39ºC. About 200 mg of the dried ground sample of each waste was incubated at 39ºC for 24 h in triplicate in 100 mL calibrated glass syringes (Haberle Labortechnik, Germany) with buffered rumen fluid for assessing the net gas production, ME availability, true organic matter (OM) and NDF digestibility (Menke et al., 1979; Menke and Steingass, 1988; Hundal et al., 2019) [18,19] . The microbial biomass production (MBP) was estimated according to Grings et al. (2005) [9] . MBP = (IVTDOM, mg/g) -(net gas production, ml/g) × 2.2 The metabolizable energy (ME) was estimated as per Menke et al. (1979) formula [19]: ME (MJ/kg DM) = 1.54 + 0.145 × net gas production (ml/200 mg DM) + 4.12 × CP (g/g DM) + 6.5 × CP (g/gDM) × CP (g/g) + 20.6 × ether extract (g/g DM) (Shilwant et al., 2022) [28] .

Statistical analysis

Data were analysed by using one-way ANOVA with inclusion of bakery waste as the fixed effect. Tukey's test used for comparing mean of different treatment. Linear and quadratic effects of bakery waste inclusion were evaluated using orthogonal polynomial contrast. Significant difference were considered at P < 0.05. Statistical analyses were done by using SPSS 20.0.

Result

The biochemical characterization of bakery waste (on a dry matter basis) is presented in Table 1 4. The net gas production was 264 ml/g at 24 h with 96.6% in vitro true organic matter degradability (IVTOMD), 66.0% in vitro neutral detergent fibre degradability (IVNDFD), while metabolizable energy (ME) was 13.0 MJ/kg and microbial biomass production (MBP) 731 mg/g digested OM. The effect of the inclusion of Bakery waste on biochemical evaluation of in vitro fermentation is presented in Table 5. As the inclusion of bakery waste increased from 0 to 40%, a significant linear improvement (P<0.001) was reported in all in vitro fermentation parameters except microbial biomass production (MBP). NGP increased from 159.2 ml/g at 0% inclusion to 182.5 ml/g at 40% inclusion (P < 0.001), with significant difference observed between the 20% to 40% inclusion levels. A significant difference was observed in IVNDFD (P = 0.003) and IVTOMD (P < 0.05) from 25 to 40% inclusion of bakery waste. ME increased from 9.20 MJ/kg at 0% inclusion to 11.1 MJ/kg at 40% inclusion (P < 0.001), with significant differences observed across all inclusion levels. Microbial biomass production (MBP) ranged from 549.4 to 681 mg/g digested OM, remains unaffected (P > 0.05) across different levels of bakery waste.

Discussion

Biochemical composition of bakery waste and experimental diets

The result of high starch, sugar and ether-extract content of bakery waste reported in the present study is aligned with previous literature, which classified bakery waste as energyrich feed ingredients (El-Haysha et al., 2017) [6] . High energy content (ME and NE L ) in bakery waste was observed due to the addition of oil and butter during the processing of the product. A similar high NE L value (9.3 MJ/ kg of DM) for bakery by-products was observed in a previous study of Humer et al. (2018) [11] . The results for sugar, starch and NDF are closely matched with Nayohan et al. (2024) [22] , who reported that bakery by-products contain sugar, starch and NDF at 210 to 590 g/kg DM, 260 to 320 g/kg DM and 15 to 43 g/kg DM, respectively. Similarly, Humer et al.

(2018) [11] also noted the low NDF and ADF levels in bakery by-products. In addition to this, microbiological and toxicological parameters were measured for bakery waste with total viable counts (7.8 × 10³ cfu/g) and aflatoxin (8.8 ppb) remains within safe limit, which is very essential because high moisture in industrial by-products which lead to susceptible for rapid fungal deterioration and mycotoxin contamination (Kilama et al., 2023) [15] .

In study of Humer et al. (2018) [11] , increase inclusion of bakery by-products from 0 to 45%, increased ether extract content from 2.5 to 7.1% while decreasing NDF from 34. [3,7,22,26] .

Biochemical evaluation of in vitro fermentation

The linear improvement in nutrient digestibility and increase gas production of bakery waste due to gelatinization of starch (Han et al. 2022; Nayohan et al., 2024) [10,22] . Additionally, high metabolizable energy (13.0 MJ/kg) with microbial biomass production of 731 mg/g digested OM of bakery waste indicates it high energy and high digestible feed ingredient.

As the inclusion level from 20% to 40% of bakery waste increased, significant difference was noted in net gas production (NGP) at 24 hrs. Increase utilization of easily fermentable substrate during in vitro fermentation causes the highest net gas production as seen at 40% inclusion levels (Kilama et al., 2023) [15] . A similar result of linear increase in gas production was reported in various studies ( [11,14,22] . In cereal grains, starch is enclosed within a protein matrix and a crystalline granule structure that limited the amylolytic process which cause slower fermentation lead to lower NGP (Pastorelli et al., 2023) [25] , whereas the starch in bakery products undergoes gelatinization during baking, which disrupts the granule and increases its susceptibility to microbial and enzymatic attack (Han et al., 2022) [10] . In addition to gelatinized starch, the sugar fraction of bakery waste also contribute to gas production, since simple sugars are fermented several-fold faster than starch in the rumen, with reported fractional degradation rates of approximately 300%/h for sugar compared with only 5-50%/h for starch (Nayohan et al., 2024) [22] . Additionally, Dong et al. (2021) [5] also reported that increasing dietary sugar concentration significantly increased gas production independent of starch levels. Additionally, increase in IVTOMD and IVNDFD with increasing bakery waste level indicates that the improved fermentability. Gelatinized starch is degraded more rapidly by amylolytic bacteria resulting increase in microbial fermentative activity and provide more favorable environment for fibrolytic population. In contrast to this, Firkins et al. (2025) [8] reported that high concentration of starch was suppress the fiber digestibility and reduce ruminal pH lead to acidosis conditions. In present study, this inhibitory effect did not appear to occur in the fiber digestibility mainly due to very low level of NDF and ADF in bakery waste which significantly reduce load of fiber and prevent acidosis conditions. This is supported by Nayohan et al. (2024) [22] , who reported that the low residual fiber and high gelatinized-starch content of bakery by-products favored rather than compromised fiber disappearance in vitro.

The [7,13] . The abundance of rapidly fermentable substrates likely stimulated microbial activity and fermentation, thereby offsetting any potential inhibitory effects of the lipid fraction. Consequently, organic matter and fibre degradability remained unaffected.

Acknowledgement

AcknowledgmentsThe authors are thankful to the Department of Animal Nutrition, College of Veterinary Science (COVS), Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab, India, for providing facility for the conduct of this experiment. There is no conflict of interest related to this study. The presented data in this manuscript are original and have not been published elsewhere. The authors declare that no funds or other support were received during the preparation of this manuscript.

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